Cyclin D polynucleotides polypeptides and uses thereof

ABSTRACT

The invention provides isolated polynucleotides and their encoded proteins that are involved in cell cycle regulation. The invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions. The present invention provides methods and compositions relating to altering cell cycle protein content and/or composition of plants.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. patent application Ser.No. 60/101,551, filed Sep. 23,1998, to co-pending U.S. patentapplication Ser. No. 09/398,858, filed Sep. 20, 1999, and to co-pendingU.S. patent application Ser. No. 10/320,230, filed Dec. 16, 2002, whichare incorporated by reference herewithin in their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to plant molecularbiology. More specifically, it relates to nucleic acids and methods formodulating their expression in plants.

BACKGROUND OF THE INVENTION

[0003] Cell division plays a crucial role during all phases of plantdevelopment. The continuation of organogenesis and growth responses to achanging environment requires precise spatial, temporal anddevelopmental regulation of cell division activity in meristems (and incells with the capability to form new meristems such as in lateral rootformation). Such control of cell division is also important in organsthemselves (i.e. separate from meristems per se), for example, in leafexpansion, secondary growth, and endoreduplication.

[0004] A complex network controls cell proliferation in eukaryotes.Regulatory pathways communicate environmental constraints, such asnutrient availability, mitogenic signals such as growth factors orhormones, or developmental cues such as the transition from vegetativeto reproductive. Ultimately, these regulatory pathways control thetiming, frequency (rate), plane and position of cell divisions.

[0005] The basic mechanism of cell cycle control is conserved amongeukaryotes. A catalytic protein serine/threonine kinase and anactivating cyclin subunit control progress through the cell cycle. Theprotein kinase is generally referred to as a cyclin-dependent-kinase(CDK), whose activity is modulated by phosphorylation anddephosphorylation events and by their association with regulatorysubunits called cyclins. CDKs require association with cyclins foractivation, and the timing of activation is largely dependent uponcyclin expression. CDKs are a family of serine/threonine protein kinasesthat regulate individual cell cycle transitions.

[0006] Eukaryote genomes typically encode multiple cyclin and CDK genes.In higher eukaryotes, different members of the CDK family act indifferent stages of the cell cycle. Cyclin genes are classifiedaccording to the timing of their appearance during the cell cycle. Inaddition to cyclin and CDK subunits, CDKs are often physicallyassociated with other proteins that alter localization, substratespecificity, or activity. A few examples of such CDK interactingproteins are the CDK inhibitors, members of theRetinoblastoma-associated protein (Rb) family, and the ConstitutiveKinase Subunit (CKS).

[0007] The protein kinase activity of the complex is regulated byfeedback control at certain checkpoints. At such checkpoints the CDKactivity becomes limiting for further progress. When the feedbackcontrol network senses the completion of a checkpoint, CDK is activatedand the cell passes through to the next checkpoint. Changes in CDKactivity are regulated at multiple levels, including reversiblephosphorylation of the cell cycle factors, changes in subcellularlocalization of the complex, and the rates of synthesis and destructionof limiting components. P. W. Doerner, Cell Cycle Regulation in Plants,Plant Physiol. (1994)106:823-827.

[0008] Plants have unique developmental features that distinguish themfrom other eukaryotes. Plant cells do not migrate, and thus only celldivision, expansion and programmed cell death determine morphogenesis.Organs are formed throughout the entire life span of the plant fromspecialized regions called meristems. In addition, many differentiatedcells have the potential to both dedifferentiate and to reenter the cellcycle. There are also numerous examples of plant cell types that undergoendoreduplication, a process involving nuclear multiplication withoutcytokinesis. The study of plant cell cycle control genes is expected tocontribute to the understanding of these unique phenomena. O. Shaul etal., Regulation of Cell Division in Arabidopsis, Critical Reviews inPlant Sciences, 15(2):97-112 (1996).

[0009] There is evidence to suggest that cells must be dividing fortransformation to occur. It has also been observed that dividing cellsrepresent only a fraction of cells that transiently express a transgene.Furthermore, the presence of damaged DNA in non-plant systems (similarto DNA introduced by particle gun or other physical means) has been welldocumented to rapidly induce cell cycle arrest (W. Siede, Cell cyclearrest in response to DNA damage: lessons from yeast, Mutation Res.337(2):73-84). Therefore, to optimize transformation it would bedesirable to provide a method for increasing the number of cellsundergoing division.

[0010] Cell division in higher eukaryotes is controlled by two maincheckpoints in the cell cycle that prevent the cell from entering eitherM- or S-phase of the cycle prematurely. Evidence from yeast andmammalian systems has repeatedly shown that over-expression of key cellcycle activating genes can either trigger cell division in non-dividingcells, or stimulate division in previously dividing cells (i.e. theduration of the cell cycle is decreased and cell size is reduced).Examples of genes whose over-expression has been shown to stimulate celldivision include cyclins (see, e.g. Doerner, P. et al., Nature (1996)380:520-423; Wang, T. C., et al., Nature (1994) 369:669-671; Quelle D.E., et al., Genes Dev. (1993) 7:1559-1571, E2F transcription factors(see, e.g. Johnson D. G. et al., Nature (1993) 365:349-352; Lukas, J. etal., (1996) Mol. Cell. Biol. 16:1047-1057), cdc25 (see, e.g. Bell, M. H.et al., (1993) Plant Molecular Biology 23:445-451; Draetta, D. et al.,(1996) BBA 1332:53-63), and mdm2 (see, e.g. Teoh, G. et al., (1997)Blood 90:1982-1992). Conversely, other gene products have been found toparticipate in negative regulation and/or checkpoint control,effectively blocking or retarding progression through the cell cycle.(see MacLachlan, T. K. et al., (1995) Critical Rev. Eukaroytic GeneExpression 5(2):127-156).

[0011] Current methods for genetic engineering in maize require aspecific cell type as the recipient of new DNA. These cells are found inrelatively undifferentiated, rapidly growing callus cells or on thescutellar surface of the immature embryo (which gives rise to callus).Irrespective of the delivery method currently used, DNA is introducedinto literally thousands of cells, yet transformants are recovered atfrequencies of 10⁻⁵ relative to transiently-expressing cells.Exacerbating this problem, the trauma that accompanies DNA introductiondirects recipient cells into cell cycle arrest and accumulating evidencesuggests that many of these cells are directed into apoptosis orprogrammed cell death. (Bowen et al., Tucson International Mol. Biol.Meetings).

[0012] Over the period between 1950 and 1980, the increase in maizeproduction worldwide outpaced both wheat and rice. Despite a temporarydownswing in the early to mid-1980's (due to both environmental andpolitical factors) world maize production has risen steadily from around145 million tons in 1950 to nearly 500 million tons by 1990. Increasesin yield and harvested area have been the predominant contributors toenhanced world production; with yield playing the major role inindustrialized countries and area expansion being most important indeveloping countries. Yet, over the next ten years it's also predictedthat meeting the demand for corn worldwide will require an additional20% over current production (Dowswell, C. R., Paliwal, R. L., Cantrell,R. P. (1996) Maize in the Third World, Westview Press, Boulder, Colo.).

[0013] The components most often associated with maize productivity aregrain yield or whole-plant harvest for animal feed (in the forms ofsilage, fodder, or stover). Thus the relative growth of the vegetativeor reproductive organs might be preferred, depending on the ultimate useof the crop. Whether the whole plant or the ear are harvested, overallyield will depend strongly on vigor and growth rate. In modern maizehybrids, the impact of heterosis on overall plant vigor and yield hasbeen unarguably demonstrated (Duvick, D. N. (1984) In: Geneticcontributions to yield gains in five major crop plants. W. R. Fehr, ed.CSSA, Madison, Wis.). Corn breeders since the 1930's have beenselectively breeding by identifying inbreds that in combination producehybrid vigor well beyond either parent. Surprisingly little is knownabout why hybrids are so much larger than their parent inbreds, althoughthere are some interesting observations in the literature. In metabolicstudies, heterosis (increases over either parent) has been observed forphysiological traits such as P uptake by roots (Baliger and Barber,1979; Nielsen and Barber, 1978), but for many enzymatic traits thehybrid is often intermediate to the inbred parents (Hageman, R. H.,Leng, E. R., Dudley, J. W. (1967) Adv. Agron. 19:45-86; Chevalier, P.,Schrader, L. E. (1977) Crop Sci. 17:897-901; Schrader, L. E. (1974) CropSci. 14:201-205; Schrader, L. E. (1985) pp 79-89. In: Exploitation ofphysiological and genetic variability to enhance crop productivity.Harper, J. E. ed. Am. Soc. Plant Physiol. Rockville, Md., Schrader, L.E., Cataldo, D. A., Peterson, D. M., Vogelzang, R. D. (1974) PlantPhysiol. 32:337-341).

[0014] Anatomical data is less confusing. In summarizing data from anearlier publication, Kiesselbach states that approximately 10% of theincreased vigor of the hybrid over its inbred parents is due to cellenlargement, and 90% can be accounted for simply by increased cellnumbers (Kiesselbach, T. A. 1922,1949. The Structure and Reproduction ofCorn, Nebraska Agric. Exp. Stn. Res. Bull. 161). This evidence forenhanced cell divisions contributing to increased maize vigor remainsunchallenged. Recently it was shown that overexpressing a B cyclin inArabidopsis resulted in increased root biomass and the root cells weresmaller (indicative of accelerated cell division), but the overall plantmorphology was not perturbed (Doerner et al., 1996). Similarly,expression of maize CycD genes in corn will enhance growth and biomassaccumulation.

[0015] Other more specialized applications exist for these genes at thewhole plant level. It has been demonstrated that endoreduplicationoccurs in numerous cell types within plants, but this is particularlyprevalent in maize endosperm, the primary seed storage tissue. Under thedirection of endosperm-specific promoters, expression of CycD genes (andpossibly expression of CycD in conjunction with genes that inhibitmitosis) will further stimulate the process of endoreduplication.

SUMMARY OF THE INVENTION

[0016] Generally, it is the object of the present invention to providenucleic acids and proteins relating to the control of cell division.

[0017] It is another object of the present invention to provide nucleicacids and proteins that can be used to identify other interactingproteins involved in cell cycle regulation.

[0018] It is another object of the present invention to provideantigenic fragments of the proteins of the present invention.

[0019] It is another object of the present invention to providetransgenic plants comprising the nucleic acids of the present invention.

[0020] It is another object of the present invention to provide methodsfor modulating, in a transgenic plant, the expression of the nucleicacids of the present invention.

[0021] It is another object of the present invention to provide a methodfor increasing the number of cells undergoing cell division.

[0022] It is another object of the present invention to provide a methodfor increasing crop yield.

[0023] It is another object of the present invention to provide a methodfor improving transformation frequencies.

[0024] It is another object of the present invention to provide a methodfor providing a positive growth advantage in a plant comprisingmodulating CycD protein expression.

[0025] It is another object of the present invention to provide a methodfor modulating cell growth.

[0026] It is another object of the present invention to provide a methodfor modulating cell division.

[0027] It is another object of the present invention to provide a methodfor modulating plant height or size.

[0028] It is another object of the present invention to provide a methodfor providing a positive growth advantage.

[0029] It is another object of the present invention to provide a methodfor increasing the growth rate.

[0030] It is another object of the present invention to provide a methodfor enhancing or inhibiting organ growth, for example seed, root, shoot,ear, tassel, stalk, pollen, stamen.

[0031] It is another object of the present invention to provide a methodfor producing organ ablation.

[0032] It is another object of the present invention to provide a methodfor producing parthenocarpic fruits.

[0033] It is another object of the present invention to provide a methodfor producing male sterile plants.

[0034] It is another object of the present invention to provide a methodfor enhancing embryogenic response, i.e. size or growth rate.

[0035] It is another object of the present invention to provide a methodfor increasing callus induction.

[0036] It is another object of the present invention to provide a methodfor positive selection.

[0037] It is another object of the present invention to provide a methodfor increasing plant regeneration.

[0038] It is another object of the present invention to provide a methodfor altering the percent of time that cells are arrested, i.e. in G1 orG0 stages of the cell cycle.

[0039] It is another object of the present invention to provide a methodfor altering the amount of time a cell spends in a particular cellcycle.

[0040] It is another object of the present invention to provide a methodfor improving in cells the response to environmental stress such asdrought, heat, or cold.

[0041] It is another object of the present invention to provide a methodfor increasing the number of pods per plant.

[0042] It is another object of the present invention to provide a methodfor increasing the number of seeds per pod or ear.

[0043] It is another object of the present invention to provide a methodfor altering the lag time in seed development.

[0044] It is another object of the present invention to provide a methodfor providing hormone independent cell growth.

[0045] It is another object of the present invention to provide a methodfor increasing growth rate of cells in bioreactors.

[0046] Therefore, in one aspect, the present invention relates to anisolated nucleic acid comprising a member selected from the groupconsisting of:

[0047] (a) a polynucleotide that encodes a polypeptide of SEQ ID NOS: 1,11, 13, or 21;

[0048] (b) a polynucleotide amplified from a monocot nucleic acidlibrary using the primers of SEQ ID NOS: 3-10, 15-20 or 23-30;

[0049] (c) a polynucleotide having 20 contiguous bases of SEQ ID NOS: 1,11, 13, or21;

[0050] (d) a polynucleotide encoding a monocot cyclin D protein;

[0051] (e) a polynucleotide having at least 70% identity to the entirecoding region of SEQ ID NOS: 1, 11, 13, or 21, wherein the % identity isdetermined by GCG/bestfit program using a gap creation penalty of 50 anda gap extension penalty of 3;

[0052] (f) a polynucleotide that hybridizes under stringent conditionsto a nucleic acid characterized by SEQ ID NOS: 1, 11, 13, or 21, whereinthe conditions include a wash in 0.1×SSC at 60 to 65° C.;

[0053] (g) a polynucleotide characterized by the sequences set forth inSEQ ID NOS: 1, 11, 13, or 21;

[0054] (h) An isolated nucleic acid amplified from a Zea mays nucleicacid library using the primers of SEQ ID NOS: 3-10,15-20 or 23-30;

[0055] (i) a polynucleotide complementary to a polynucleotide of (a)through (g); and

[0056] (j) a polynucleotide having the sequence of ATCC deposit havingthe Designation No. 98847 or 98848.

[0057] In another aspect, the present invention relates to recombinantexpression cassettes, comprising the nucleic acid operably linked to apromoter.

[0058] In some embodiments, the nucleic acid is operably linked inantisense orientation to the promoter.

[0059] In another aspect, the present invention is directed to a hostcell transfected with the recombinant expression cassette as described,supra.

[0060] In a further aspect, the present invention relates to an isolatedprotein comprising a polypeptide of at least 10 contiguous amino acidsencoded by the isolated nucleic acid. In some embodiments, thepolypeptide has a sequence selected from the group consisting of SEQ IDNOS: 2, 12, 14, and 22.

[0061] In another aspect, the present invention relates to an isolatednucleic acid comprising a polynucleotide of at least 25 nucleotides inlength which selectively hybridizes under stringent conditions to anucleic acid selected from the group consisting of SEQ ID NOS: 1, 11,13, and 21, or a complement thereof. In some embodiments, the isolatednucleic acid is operably linked to a promoter.

[0062] In yet another aspect, the present invention relates to anisolated nucleic acid comprising a polynucleotide, the polynucleotidehaving at least 80% sequence identity to an identical length of anucleic acid selected from the group consisting of SEQ ID NOS: 1, 11,13, and 21 or a complement thereof.

[0063] In another aspect, the present invention relates to an isolatednucleic acid comprising a polynucleotide having a sequence of a nucleicacid amplified from a Zea mays nucleic acid library using the primersselected from the group consisting of SEQ ID NOS: 3-10, 15-20, and 23-30or complements thereof. In some embodiments, the nucleic acid library isa cDNA library.

[0064] In another aspect, the present invention relates to a recombinantexpression cassette comprising a nucleic acid amplified from a libraryas referred to supra, wherein the nucleic acid is operably linked to apromoter.

[0065] In some embodiments, the present invention relates to a host celltransfected with this recombinant expression cassette.

[0066] In some embodiments, the present invention relates to a proteinof the present invention that is produced from this host cell.

[0067] In an additional aspect, the present invention is directed to anisolated nucleic acid comprising a polynucleotide encoding a polypeptidewherein: (a) the polypeptide comprises at least 10 contiguous amino acidresidues from a first polypeptide selected from the group consisting ofSEQ ID NOS: 2,12, 14, and 22; (b) the polypeptide does not bind toantisera raised against the first polypeptide which has been fullyimmunosorbed with the first polypeptide; and (c) the polypeptide has amolecular weight in non-glycosylated form within 10% of the firstpolypeptide.

[0068] In a further aspect, the present invention relates to aheterologous promoter operably linked to a non-isolated polynucleotideof the present invention, wherein the polypeptide is encoded by anucleic acid amplified from a nucleic acid library.

[0069] In yet another aspect, the present invention relates to atransgenic plant comprising a recombinant expression cassette comprisinga plant promoter operably linked to any of the isolated nucleic acids ofthe present invention. The present invention also provides transgenicseed from the transgenic plant.

[0070] In a further aspect, the present invention relates to a method ofmodulating expression of the genes-encoding the proteins of the presentinvention in a plant, comprising the steps of (a) transforming a plantcell with a recombinant expression cassette comprising a polynucleotideof the present invention operably linked to a promoter; (b) growing theplant cell under plant growing conditions; and (c) inducing expressionof the polynucleotide for a time sufficient to modulate expression ofthe genes in the plant. Expression of the genes encoding the proteins ofthe present invention can be increased or decreased relative to anon-transformed control plant.

[0071] In another aspect of the invention an isolated protein isprovided comprising a member selected from the group consisting of:

[0072] (a) a polypeptide comprising at least 25 contiguous amino acidsof SEQ ID NOS: 2, 12, 14, or 22;

[0073] (b) a polypeptide which is a monocot cyclin D protein;

[0074] (c) a polypeptide comprising at least 65% sequence identity toSEQ ID NOS: 2, 12, 14, or 22, wherein the % sequence identity is basedon the entire sequence and is determined by GAP 10 using defaultparameters;

[0075] (d) a polypeptide encoded by a nucleic acid of claim 1; and

[0076] (e) a polypeptide characterized by SEQ ID NO: 2, 12, 14, or 22.

BRIEF DESCRIPTION OF THE DRAWINGS

[0077]FIGS. 1 and 2 represent transformation frequency in treatmentscontaining the ZmCycD gene compared to transformation without ZmCycD.

DEFINITIONS

[0078] Units, prefixes, and symbols may be denoted in their Si acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric ranges areinclusive of the numbers defining the range. Amino acids may be referredto herein by either their commonly known three letter symbols or by theone-letter symbols recommended by the IUPAC-IUB Biochemical NomenclatureCommission. Nucleotides, likewise, may be referred to by their commonlyaccepted single-letter codes. The terms defined below are more fullydefined by reference to the specification as a whole.

[0079] By “amplified” is meant the construction of multiple copies of anucleic acid sequence or multiple copies complementary to the nucleicacid sequence using at least one of the nucleic acid sequences as atemplate. Amplification systems include the polymerase chain reaction(PCR) system, ligase chain reaction (LCR) system, nucleic acid sequencebased amplification (NASBA, Cangene, Mississauga, Ontario), Q-BetaReplicase systems, transcription-based amplification system (TAS), andstrand displacement amplification (SDA). See, e.g., Diagnostic MolecularMicrobiology: Principles and Applications, D. H. Persing et al., Ed.,American Society for Microbiology, Washington, D.C. (1993). The productof amplification is termed an amplicon.

[0080] The term “antibody” includes reference to antigen binding formsof antibodies (e.g., Fab, F(ab)₂). The term “antibody” frequently refersto a polypeptide substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, which specifically bind andrecognize an analyte (antigen). However, while various antibodyfragments can be defined in terms of the digestion of an intactantibody, one of skill will appreciate that such fragments may besynthesized de novo either chemically or by utilizing recombinant DNAmethodology. Thus, the term antibody, as used herein, also includesantibody fragments such as single chain Fv, chimeric antibodies (i.e.,comprising constant and variable regions from different species),humanized antibodies (i.e., comprising a complementarity determiningregion (CDR) from a non-human source) and heteroconjugate antibodies(e.g., bispecific antibodies).

[0081] The term “antigen” includes reference to a substance to which anantibody can be generated and/or to which the antibody is specificallyimmunoreactive. The specific immunoreactive sites within the antigen areknown as epitopes or antigenic determinants. These epitopes can be alinear array of monomers in a polymeric composition—such as amino acidsin a protein—or consist of or comprise a more complex secondary ortertiary structure. Those of skill will recognize that all immunogens(i.e., substance capable of eliciting an immune response) are antigens;however some antigens, such as haptens, are not immunogens but may bemade immunogenic by coupling to a carrier molecule. An antibodyimmunologically reactive with a particular antigen can be generated invivo or by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors. See, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward, et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996).

[0082] As used herein, “antisense orientation” includes reference to aduplex polynucleotide sequence that is operably linked to a promoter inan orientation where the antisense strand is transcribed. The antisensestrand is sufficiently complementary to an endogenous transcriptionproduct such that translation of the endogenous transcription product isoften inhibited.

[0083] As used herein, “chromosomal region” includes reference to alength of chromosome that can be measured by reference to the linearsegment of DNA that it comprises. The chromosomal region can be definedby reference to two unique DNA sequences, i.e., markers.

[0084] The term “conservatively modified variants” applies to both aminoacid and nucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or conservatively modified variants of theamino acid sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenprotein. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations” and represent onespecies of conservatively modified variation. Every nucleic acidsequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of ordinary skillwill recognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine) can be modified to yield afunctionally identical molecule. Accordingly, each silent variation of anucleic acid which encodes a polypeptide of the present invention isimplicit in each described polypeptide sequence and incorporated hereinby reference.

[0085] As to amino acid sequences, one of skill will recognize thatindividual substitutions, deletions or additions to a nucleic acid,peptide, polypeptide, or protein sequence which alters, adds or deletesa single amino acid or a small percentage of amino acids in the encodedsequence is a “conservatively modified variant” where the alterationresults in the substitution of an amino acid with a chemically similaramino acid. Thus, any number of amino acid residues selected from thegroup of integers consisting of from 1 to 15 can be so altered. Thus,for example, 1, 2, 3, 4, 5, 7, or 10 alterations can be made.Conservatively modified variants typically provide similar biologicalactivity as the unmodified polypeptide sequence from which they arederived. For example, substrate specificity, enzyme activity, orligand/receptor binding is generally at least 30%, 40%, 50%, 60%, 70%,80%, or 90% of the native protein for it's native substrate.Conservative substitution tables providing functionally similar aminoacids are well known in the art.

[0086] The following six groups each contain amino acids that areconservative substitutions for one another:

[0087] 1) Alanine (A), Serine (S), Threonine (T);

[0088] 2) Aspartic acid (D), Glutamic acid (E);

[0089] 3) Asparagine (N), Glutamine (Q);

[0090] 4) Arginine (R), Lysine (K);

[0091] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0092] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0093] See also, Creighton (1984) Proteins W. H. Freeman and Company.

[0094] By “encoding” or “encoded”, with respect to a specified nucleicacid, is meant comprising the information for translation into thespecified protein. A nucleic acid encoding a protein may comprisenon-translated sequences (e.g., introns) within translated regions ofthe nucleic acid, or may lack such intervening non-translated sequences(e.g., as in cDNA). The information by which a protein is encoded isspecified by the use of codons. Typically, the amino acid sequence isencoded by the nucleic acid using the “universal” genetic code. However,variants of the universal code, such as is present in some plant,animal, and fungal mitochondria, the bacterium Mycoplasma capricolum(Proc. Natl. Acad. Sci., U.S.A. 82:2306-2309 (1985)), or the ciliateMacronucleus, may be used when the nucleic acid is expressed using theseorganisms.

[0095] When the nucleic acid is prepared or altered synthetically,advantage can be taken of known codon preferences of the intended hostwhere the nucleic acid is to be expressed. For example, although nucleicacid sequences of the present invention may be expressed in bothmonocotyledonous and dicotyledonous plant species, sequences can bemodified to account for the specific codon preferences and GC contentpreferences of monocotyledons or dicotyledons as these preferences havebeen shown to differ (Murray et al., Nucl. Acids Res. 17:477-498(1989)). Thus, the maize preferred codon for a particular amino acid canbe derived from known gene sequences from maize. Maize codon usage for28 genes from maize plants are listed in Table 4 of Murray et al.,supra.

[0096] As used herein “full-length sequence” in reference to a specifiedpolynucleotide or its encoded protein means having the entire amino acidsequence of, a native (non-synthetic), endogenous, catalytically activeform of the specified protein. A full-length sequence can be determinedby size comparison relative to a control that is a native(non-synthetic) endogenous cellular form of the specified nucleic acidor protein. Methods to determine whether a sequence is full-length arewell known in the art including such exemplary techniques as northern orwestern blots, primer extension, S1 protection, and ribonucleaseprotection. See, e.g., Plant Molecular Biology: A Laboratory Manual,Clark, Ed., Springer-Verlag, Berlin (1997). Comparison to knownfull-length homologous (orthologous and/or paralogous) sequences canalso be used to identify full-length sequences of the present invention.Additionally, consensus sequences typically present at the 5′ and 3′untranslated regions of mRNA aid in the identification of apolynucleotide as full-length. For example, the consensus sequenceANNNNAUGG, where the underlined codon represents the N-terminalmethionine, aids in determining whether the polynucleotide has acomplete 5′ end. Consensus sequences at the 3′ end, such aspolyadenylation sequences, aid in determining whether the polynucleotidehas a complete 3′ end.

[0097] As used herein, “heterologous” in reference to a nucleic acid isa nucleic acid that originates from a foreign species, or, if from thesame species, is substantially modified from its native form incomposition and/or genomic locus by deliberate human intervention. Forexample, a promoter operably linked to a heterologous structural gene isfrom a species different from that from which the structural gene wasderived, or, if from the same species, one or both are substantiallymodified from their original form. A heterologous protein may originatefrom a foreign species or, if from the same species, is substantiallymodified from its original form by deliberate human intervention.

[0098] By “host cell” is meant a cell that contains a vector andsupports the replication and/or expression of the expression vector.Host cells may be prokaryotic cells such as E. coli, or eukaryotic cellssuch as yeast, insect, amphibian, or mammalian cells. Preferably, hostcells are monocotyledonous or dicotyledenous plant cells. A particularlypreferred monocotyledonous host cell is a maize host cell.

[0099] The term “hybridization complex” includes reference to a duplexnucleic acid structure formed by two single-stranded nucleic acidsequences selectively hybridized with each other.

[0100] By “immunologically reactive conditions” or “immunoreactiveconditions” is meant conditions which allow an antibody, generated to aparticular epitope, to bind to that epitope to a detectably greaterdegree (e.g., at least 2-fold over background) than the antibody bindsto substantially all other epitopes in a reaction mixture comprising theparticular epitope. Immunologically reactive conditions are dependentupon the format of the antibody binding reaction and typically are thoseutilized in immunoassay protocols. See Harlow and Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions.

[0101] The term “introduced” in the context of inserting a nucleic acidinto a cell, means “transfection” or “transformation” or “transduction”and includes reference to the incorporation of a nucleic acid into aeukaryotic or prokaryotic cell where the nucleic acid may beincorporated into the genome of the cell (e.g., chromosome, plasmid,plastid or mitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

[0102] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with it as found in itsnaturally occurring environment. The isolated material optionallycomprises material not found with the material in its naturalenvironment; or (2) if the material is in its natural environment, thematerial has been synthetically (non-naturally) altered by deliberatehuman intervention to a composition and/or placed at a locus in the cell(e.g., genome or subcellular organelle) not native to a material foundin that environment. The alteration to yield the synthetic material canbe performed on the material within or removed from its natural state.For example, a naturally occurring nucleic acid becomes an isolatednucleic acid if it is altered, or if it is transcribed from DNA that hasbeen altered, by non-natural, synthetic (i.e., “man-made”) methodsperformed within the cell from which it originates. See, e.g., Compoundsand Methods for Site Directed Mutagenesis in Eukaryotic Cells, Kmiec,U.S. Pat. No. 5,565,350; In Vivo Homologous Sequence Targeting inEukaryotic Cells; Zarling et al., PCT/US93/03868. Likewise, a naturallyoccurring nucleic acid (e.g., a promoter) becomes isolated if it isintroduced by non-naturally occurring means to a locus of the genome notnative to that nucleic acid. Nucleic acids that are “isolated” asdefined herein, are also referred to as “heterologous” nucleic acids.

[0103] Unless otherwise stated, the term “cell cycle nucleic acid” meansa nucleic acid comprising a polynucleotide (“cell cycle polynucleotide”)encoding a cell cycle polypeptide. A “cell cycle gene” refers to anon-heterologous genomic form of a full-length cell cyclepolynucleotide.

[0104] As used herein, “localized within the chromosomal region definedby and including” with respect to particular markers includes referenceto a contiguous length of a chromosome delimited by and including thestated markers.

[0105] As used herein, “marker” includes reference to a locus on achromosome that serves to identify a unique position on the chromosome.A “polymorphic marker” includes reference to a marker which appears inmultiple forms (alleles) such that different forms of the marker, whenthey are present in a homologous pair, allow transmission of each of thechromosomes in that pair to be followed. A genotype may be defined byuse of one or a plurality of markers.

[0106] As used herein, “nucleic acid” includes reference to adeoxyribonucleotide or ribonucleotide polymer in either single- ordouble-stranded form, and unless otherwise limited, encompasses knownanalogues having the essential nature of natural nucleotides in thatthey hybridize to single-stranded nucleic acids in a manner similar tonaturally occurring nucleotides (e.g., peptide nucleic acids).

[0107] By “nucleic acid library” is meant a collection of isolated DNAor RNA molecules that comprise and substantially represent the entiretranscribed fraction of a genome of a specified organism. Constructionof exemplary nucleic acid libraries, such as genomic and cDNA libraries,is taught in standard molecular biology references such as Berger andKimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology,Vol. 152, Academic Press, Inc., San Diego, Calif. (Berger); Sambrook etal., Molecular Cloning—A Laboratory Manual, 2nd ed., Vol. 1-3 (1989);and Current Protocols in Molecular Biology, F. M. Ausubel et al., Eds.Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc. (1994 Supplement).

[0108] As used herein “operably linked” includes reference to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand, where necessary to join two protein coding regions, contiguous andin the same reading frame.

[0109] As used herein, the term “plant” includes reference to wholeplants, plant organs (e.g., leaves, stems, roots, etc.), seeds and plantcells and progeny of same. Plant cell, as used herein includes, withoutlimitation, seeds suspension cultures, embryos, meristematic regions,callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen,and microspores. The class of plants which can be used in the methods ofthe invention is generally as broad as the class of higher plantsamenable to transformation techniques, including both monocotyledonousand dicotyledonous plants. A particularly preferred plant is Zea mays.

[0110] As used herein, “polynucleotide” includes reference to adeoxyribopolynucleotide, ribopolynucleotide, or analogs thereof, thathave the essential nature of a natural ribonucleotide in that theyhybridize to nucleic acids in a manner similar to naturally occurringnucleotides. A polynucleotide can be full-length or a subsequence of anative or heterologous structural or regulatory gene. Unless otherwiseindicated, the term includes reference to the specified sequence as wellas the complementary sequence thereof. Thus, DNAs or RNAs with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover, DNAs or RNAs comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically, enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including inter alia, simple andcomplex cells.

[0111] The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The essential nature of such analogues of naturally occurringamino acids is that, when incorporated into a protein, that protein isspecifically reactive to antibodies elicited to the same protein butconsisting entirely of naturally occurring amino acids. The terms“polypeptide”, “peptide” and “protein” are also inclusive ofmodifications including, but not limited to, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation. Exemplary modifications aredescribed in most basic texts, such as, Proteins—Structure and MolecularProperties, 2nd ed., T. E. Creighton, W. H. Freeman and Company, NewYork (1993). Many detailed reviews are available on this subject, suchas, for example, those provided by Wold, F., Posttranslational ProteinModifications: Perspectives and Prospects, pp.1-12 in PosttranslationalCovalent Modification of Proteins, B. C. Johnson, Ed., Academic Press,New York (1983); Seifter et al., Meth. Enzymol. 182:626-646 (1990) andRattan et al., Protein Synthesis: Posttranslational Modifications andAging, Ann. N.Y. Acad. Sci. 663:48-62 (1992). It will be appreciated, asis well known and as noted above, that polypeptides are not alwaysentirely linear. For instance, polypeptides may be branched as a resultof ubiquitination, and they may be circular, with or without branching,generally as a result of posttranslation events, including naturalprocessing event and events brought about by human manipulation which donot occur naturally. Circular, branched and branched circularpolypeptides may be synthesized by non-translation natural process andby entirely synthetic methods, as well. Modifications can occur anywherein a polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. In fact, blockage of theamino or carboxyl group in a polypeptide, or both, by a covalentmodification, is common in naturally occurring and syntheticpolypeptides and such modifications may be present in polypeptides ofthe present invention, as well. For instance, the amino terminal residueof polypeptides made in E. coil or other cells, prior to proteolyticprocessing, almost invariably will be N-formylmethionine. Duringpost-translational modification of the peptide, a methionine residue atthe NH₂-terminus may be deleted. Accordingly, this inventioncontemplates the use of both the methionine-containing and themethionine-less amino terminal variants of the protein of the invention.In general, as used herein, the term polypeptide encompasses all suchmodifications, particularly those that are present in polypeptidessynthesized by expressing a polynucleotide in a host cell.

[0112] As used herein “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Exemplary plant promoters include, but are not limited to,those that are obtained from plants, plant viruses, and bacteria whichcomprise genes expressed in plant cells such Agrobacterium or Rhizobium.Examples of promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such asleaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma.Such promoters are referred to as “tissue preferred”. Promoters thatinitiate transcription only in certain tissue are referred to as “tissuespecific”. A “cell type” specific promoter primarily drives expressionin certain cell types in one or more organs, for example, vascular cellsin roots or leaves. An “inducible” promoter is a promoter that is underenvironmental control. Examples of environmental conditions that mayeffect transcription by inducible promoters include anaerobic conditionsor the presence of light. Tissue specific, tissue preferred, cell typespecific, and inducible promoters constitute the class of“non-constitutive” promoters. A “constitutive” promoter is a promoterthat is active under most environmental conditions.

[0113] The term “cell cycle polypeptide” refers to one or more aminoacid sequences, in glycosylated or non-glycosylated form, involved inthe regulation of cell division. The term is also inclusive offragments, variants, homologs, alleles or precursors (e.g.,preproproteins or proproteins) thereof. A “cell cycle protein” comprisesa cell cycle polypeptide.

[0114] As used herein “recombinant” includes reference to a cell orvector, that has been modified by the introduction of a heterologousnucleic acid or that the cell is derived from a cell so modified. Thus,for example, recombinant cells express genes that are not found inidentical form within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all as a result of deliberate humanintervention. The term “recombinant” as used herein does not encompassthe alteration of the cell or vector by naturally occurring events(e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

[0115] As used herein, a “recombinant expression cassette” is a nucleicacid construct, generated recombinantly or synthetically, with a seriesof specified nucleic acid elements that permit transcription of aparticular nucleic acid in a target cell. The recombinant expressioncassette can be incorporated into a plasmid, chromosome, mitochondrialDNA, plastid DNA, virus, or nucleic acid fragment. Typically, therecombinant expression cassette portion of an expression vectorincludes, among other sequences, a nucleic acid to be transcribed, and apromoter.

[0116] The term “residue” or “amino acid residue” or “amino acid” areused interchangeably herein to refer to an amino acid that isincorporated into a protein, polypeptide, or peptide (collectively“protein”). The amino acid may be a naturally occurring amino acid and,unless otherwise limited, may encompass known analogs of natural aminoacids that can function in a similar manner as naturally occurring aminoacids.

[0117] The term “selectively hybridizes” includes reference tohybridization, under stringent hybridization conditions, of a nucleicacid sequence to a specified nucleic acid target sequence to adetectably greater degree (e.g., at least 2-fold over background) thanits hybridization to non-target nucleic acid sequences and to thesubstantial exclusion of non-target nucleic acids. Selectivelyhybridizing sequences typically have about at least 80% sequenceidentity, preferably 90% sequence identity, and most preferably 100%sequence identity (i.e., complementary) with each other.

[0118] The term “specifically reactive”, includes reference to a bindingreaction between an antibody and a protein having an epitope recognizedby the antigen binding site of the antibody. This binding reaction isdeterminative of the presence of a protein having the recognized epitopeamongst the presence of a heterogeneous population of proteins and otherbiologics. Thus, under designated immunoassay conditions, the specifiedantibodies bind to an analyte having the recognized epitope to asubstantially greater degree (e.g., at least 2-fold over background)than to substantially all other analytes lacking the epitope which arepresent in the sample.

[0119] Specific binding to an antibody under such conditions may requirean antibody that is selected for its specificity for a particularprotein. For example, antibodies raised to the polypeptides of thepresent invention can be selected from those antibodies that arespecifically reactive with polypeptides of the present invention. Theproteins used as immunogens can be in native conformation or denaturedso as to provide a linear epitope.

[0120] A variety of immunoassay formats may be used to select antibodiesspecifically reactive with a particular protein (or other analyte). Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. SeeHarlow and Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassay formatsand conditions that can be used to determine selective reactivity.

[0121] The terms “stringent conditions” or “stringent hybridizationconditions” includes reference to conditions under which a probe willhybridize to its target sequence, to a detectably greater degree thanother sequences (e.g., at least 2-fold over background). Stringentconditions are sequence-dependent and will be different in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences can be identified which are 100%complementary to the probe (homologous probing). Alternatively,stringency conditions can be adjusted to allow some mismatching insequences so that lower degrees of similarity are detected (heterologousprobing). Generally, a probe is less than about 1000 nucleotides inlength, preferably less than 500 nucleotides in length.

[0122] Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide.

[0123] Exemplary low stringency conditions include hybridization with abuffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC =3.0 M NaCl/0.3 Mtrisodium citrate) at 50 to 55° C. Exemplary moderate stringencyconditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1%SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplaryhigh stringency conditions include hybridization in 50% formamide, 1 MNaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Generallyhybridization is conducted for a time in the range of from four tosixteen hours.

[0124] Specificity is typically the function of post-hybridizationwashes, the critical factors being the ionic strength and temperature ofthe final wash solution. For DNA-DNA hybrids, the T_(m) can beapproximated from the equation of Meinkoth and Wahl, Anal. Biochem.,138:267-284 (1984): T_(m)=81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (%form)−500/L; where M is the molarity of monovalent cations, %GC is thepercentage of guanosine and cytosine nucleotides in the DNA, % form isthe percentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs. The T_(m) is the temperature (underdefined ionic strength and pH) at which 50% of a complementary targetsequence hybridizes to a perfectly matched probe. T_(m) is reduced byabout 1° C. for each 1% of mismatching; thus, T_(m), hybridizationand/or wash conditions can be adjusted to hybridize to sequences of thedesired identity. For example, if sequences with ≧90% identity aresought, the T_(m) can be decreased 10° C. Generally, stringentconditions are selected to be about 5° C. lower than the thermal meltingpoint (T_(m)) for the specific sequence and its complement at a definedionic strength and pH. However, severely stringent conditions canutilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than thethermal melting point (T_(m)); moderately stringent conditions canutilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower thanthe thermal melting point (T_(m)); low stringency conditions can utilizea hybridization and/or wash at 11, 12, 13, 14,15, or 20° C. lower thanthe thermal melting point (T_(m)). Using the equation, hybridization andwash compositions, and desired T_(m), those of ordinary skill willunderstand that variations in the stringency of hybridization and/orwash solutions are inherently described. If the desired degree ofmismatching results in a T_(m) of less than 45° C. (aqueous solution) or32° C. (formamide solution) it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen,Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, N.Y. (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel, et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995).

[0125] As used herein, “transgenic plant” includes reference to a plantthat comprises within its genome a heterologous polynucleotide.Generally, the heterologous polynucleotide is stably integrated withinthe genome such that the polynucleotide is passed on to successivegenerations. The heterologous polynucleotide may be integrated into thegenome alone or as part of a recombinant expression cassette.“Transgenic” is used herein to include any cell, cell line, callus,tissue, plant part or plant, the genotype of which has been altered bythe presence of heterologous nucleic acid including those transgenicsinitially so altered as well as those created by sexual crosses orasexual propagation from the initial transgenic. The term “transgenic”as used herein does not encompass the alteration of the genome(chromosomal or extra-chromosomal) by conventional plant breedingmethods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

[0126] As used herein, “vector” includes reference to a nucleic acidused in transfection of a host cell and into which can be inserted apolynucleotide. Vectors are often replicons. Expression vectors permittranscription of a nucleic acid inserted therein.

[0127] The following terms are used to describe the sequencerelationships between two or more nucleic acids or polynucleotides: (a)“reference sequence”, (b) “comparison window”, (c) “sequence identity”,(d) “percentage of sequence identity”, and (e) “substantial identity”.

[0128] (a) As used herein, “reference sequence” is a defined sequenceused as a basis for sequence comparison. A reference sequence may be asubset or the entirety of a specified sequence; for example, as asegment of a full-length cDNA or gene sequence, or the complete cDNA orgene sequence.

[0129] (b) As used herein, “comparison window” means includes referenceto a contiguous and specified segment of a polynucleotide sequence,wherein the polynucleotide sequence may be compared to a referencesequence and wherein the portion of the polynucleotide sequence in thecomparison window may comprise additions or deletions (i.e., gaps)compared to the reference sequence (which does not comprise additions ordeletions) for optimal alignment of the two sequences. Generally, thecomparison window is at least 20 contiguous nucleotides in length, andoptionally can be 30, 40, 50, 100, or longer. Those of skill in the artunderstand that to avoid a high similarity to a reference sequence dueto inclusion of gaps in the polynucleotide sequence a gap penalty istypically introduced and is subtracted from the number of matches.

[0130] Methods of alignment of sequences for comparison are well knownin the art. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman, Adv.Appl. Math. 2:482 (1981); by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970); by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. 85:2444(1988); by computerized implementations of these algorithms, including,but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics,Mountain View, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in theWisconsin Genetics Software Package, Genetics Computer Group (GCG), 575Science Dr., Madison, Wis., USA; the CLUSTAL program is well describedby Higgins and Sharp, Gene 73:237-244 (1988); Higgins and Sharp, CABIOS5:151-153 (1989); Corpet, et al., Nucleic Acids Research 16:10881-90(1988); Huang, et al., Computer Applications in the Biosciences 8:155-65(1992), and Pearson, et al., Methods in Molecular Biology 24:307-331(1994). The BLAST family of programs which can be used for databasesimilarity searches includes: BLASTN for nucleotide query sequencesagainst nucleotide database sequences; BLASTX for nucleotide querysequences against protein database sequences; BLASTP for protein querysequences against protein database sequences; TBLASTN for protein querysequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel et al.,Eds., Greene Publishing and Wiley-Interscience, New York (1995).

[0131] As those of ordinary skill in the art will understand, BLASTsearches assume that proteins can be modeled as random sequences.However, many real proteins comprise regions of nonrandom sequences thatmay be homopolymeric tracts, short-period repeats, or regions enrichedin one or more amino acids. Such low-complexity regions may be alignedbetween unrelated proteins even though other regions of the protein areentirely dissimilar. A number of low-complexity filter programs can beemployed to reduce such low-complexity alignments. For example, the SEG(Wooten and Federhen, Comput. Chem., 17:149-163 (1993)) and XNU(Claverie and States, Comput. Chem., 17:191-201 (1993)) low-complexityfilters can be employed alone or in combination.

[0132] (c) As used herein, “sequence identity” or “identity” in thecontext of two nucleic acid or polypeptide sequences includes referenceto the residues in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window. Whenpercentage of sequence identity is used in reference to proteins it isrecognized that residue positions which are not identical often differby conservative amino acid substitutions, where amino acid residues aresubstituted for other amino acid residues with similar chemicalproperties (e.g. charge or hydrophobicity) and therefore do not changethe functional properties of the molecule. Where sequences differ inconservative substitutions, the percent sequence identity may beadjusted upwards to correct for the conservative nature of thesubstitution. Sequences which differ by such conservative substitutionshave “sequence similarity” or “similarity”. Means for making thisadjustment are well known to those of skill in the art. Typically thisinvolves scoring a conservative substitution as a partial rather than afull mismatch, thereby increasing the percentage sequence identity.Thus, for example, where an identical amino acid is given a score of 1and a non-conservative substitution is given a score of zero, aconservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., according tothe algorithm of Meyers and Miller, Computer Applic. Biol. Sci. 4:11-17(1988) e.g., as implemented in the program PC/GENE (Intelligenetics,Mountain View, Calif., USA).

[0133] (d) As used herein, “percentage of sequence identity” means thevalue determined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity. For purposes of defining the invention, % identity onthe nucleic acid level is determined by the BESTFIT DNA SequenceAlignment software on Genescape using a gap weight of 50 and a lengthweight of 3. For purposes of defining the invention, % identity on theamino acid level is determined by the BESTFIT DNA Sequence Alignmentsoftware on Genescape using a gap weight of 12 and a length weight of 4.

[0134] (e) (i) The term “substantial identity” of polynucleotidesequences means that a polynucleotide comprises a sequence that has atleast 70% sequence identity, preferably at least 80%, more preferably atleast 90% and most preferably at least 95%, compared to a referencesequence using one of the alignment programs described using standardparameters. One of skill will recognize that these values can beappropriately adjusted to determine corresponding identity of proteinsencoded by two nucleotide sequences by taking into account codondegeneracy, amino acid similarity, reading frame positioning and thelike. Substantial identity of amino acid sequences for these purposesnormally means sequence identity of at least 85%, more preferably atleast 90%, and most preferably at least 98%.

[0135] Another indication that nucleotide sequences are substantiallyidentical is if two molecules hybridize to each other under stringentconditions. However, nucleic acids which do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides which they encode are substantially identical. This mayoccur, e.g., when a copy of a nucleic acid is created using the maximumcodon degeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is that thepolypeptide which the first nucleic acid encodes is immunologicallycross reactive with the polypeptide encoded by the second nucleic acid.

[0136] (e) (ii) The term “substantial identity” in the context of apeptide indicates that a peptide comprises a sequence with at least 70%sequence identity to a reference sequence, preferably 80%, morepreferably 85%, most preferably at least 90% or 95% sequence identity tothe reference sequence over a specified comparison window. Optimalalignment can be conducted using the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48:443 (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution. Peptides which are “substantially similar” share sequencesas noted above except that residue positions which are not identical maydiffer by conservative amino acid changes.

[0137] By “two-hybrid system” is meant a screening method to identifyprotein-protein interactions, using a known gene (and its encodedproduct) as a “bait” or target and screening a library of expressedgenes and their corresponding encoded products for specific interactionswith the “bait” molecule. Methods for library construction and use ofvisual marker genes for yeast two-hybrid screens are well known in theart, and can be found in Sambrook, et al., 1990, Ausubel et al., 1990and G. Hannon and P. Bartel, Identification of interacting proteinsusing the two-hybrid system. Methods Mol Cellular Biol. 5:289-297(1995).

DETAILED DESCRIPTION OF THE INVENTION

[0138] The CycD genes in plants encode proteins ranging from 37 to 44kD. This protein is necessary for progression from G1 into S-phase. Theencoded protein binds to CDK4, and this active cyclin D-CDK4 kinasehyperphosphorylates Rb, releasing the E2F transcription factor whichactivates DNA synthesis. G1/S phase cyclins were first isolated in yeast(Hadwiger et al., 1989; Richardson et al., 1989), and a few years laterin humans (Matsushime et al., 1991). Subsequently, it has been cloned invarious other organisms including plants. Three CycD isoforms have beenfound in both animals and plants, which are analogous to, and cancomplement function of, the three CLN genes originally identified inyeast. In mammalian cells, cyclins appear to be important integrators ofgrowth signals for cell cycle control. In plants, this aspect has beenbest characterized in Arabidopsis, with AtCycD2 and AtCycD3 expressionbeing induced by sucrose and cytokinin, respectively (Francis et al.,1998). AtCycD3 can also been induced by nitrate levels (Fuerst et al.,1998). CycD1 has been cloned in Arabidopsis thaliana (Soni et al., 1995;EMBL accession number X83369), Antirrhinum majus and Helianthustuberosum. Cyclin D2 has been cloned in Arabidopsis (Soni et al, 1995;X83370), and CycD3 has been cloned in Arabidopsis (Soni et al., 1995;X83371), Antirrhinum, Helianthus (Freeman and Muray, unpublished),Nicotiana and Medicago (Dahl et al., 1995; X88864). No monocot homologshave been reported. In the present invention, we describe the fulllength clone of the maize CycD gene (designated ZmCycD).

[0139] In addition to the positive influence of transient cell cyclestimulation, stable expression of positive cell cycle regulators wouldbe a benefit for positive selection schemes in the recovery oftransgenic plants and plant cells. In a population of cells and/orcallus growing in vitro, cells expressing a gene such as CycD will havea differential growth advantage based simply on their accelerateddivision rate. It would be expected that these transgenic cells orcell/clusters would grow more rapidly than their non-transformedcounterparts in culture, permitting ready identification oftransformants. Such a positive growth advantage (imparted by expressionof a gene such as CycD, or CycD plus another cell cycle component),would also be beneficial in other types of transformation strategies,including as examples, protoplast transformation, leaf basetransformation and transformation of cells in meristems. Such growthstimulation may also extend transformation protocols to tissues normallyno amenable to culture. Examples would include such tissues as portionsof leaves (in which the cells do not normally divide), scutellum fromrecalcitrant inbreds (in which cells typically are not induced to dividein culture), and nodal tissues, etc.

[0140] Of particular interest is the use of cell cycle genes such asCycD to impart a positive growth advantage to cells in the meristem,including apical initials. The apical initials in angiosperm shootmeristems are defined by their position within the meristem. If anapical initial cell becomes compromised relative to neighboring cells inthe meristem, it will be replaced by an adjacent neighbor that is not ata disadvantage. This new cell assumes the role of the apical initial.Conversely, transgenic cells adjacent to the apical initials with apositive growth advantage can, over time (i.e. through successive cellgenerations), out-compete the wild-type apical initials, eventuallyreplacing these cells and establishing a homogeneous transformedmeristem.

[0141] There can also be organ and/or whole plant impacts to such cellcycle transgene expression.

[0142] References

[0143] Renaudin, J- P., Doonan, J. H., Freeman, D., Hashimoto, J., Hirt,H., Inze, D., Jacobs, T., Kouchi, H., Rouze, P., Sauter, M., Savoure,A., Sorrell, D. A., Sundaresan, V., and Murray, J. A. H. 1996. Plantcyclins: a unified nomenclature for plant A-, B- and D-type cyclinsbased on sequence organization. Plant Molecular Biology 32:1003-1018.

[0144] Dahl, M., Meskiene, I., Boegre, L., Ha, D. T. C., Swoboda, I.,Hubmann, R., Hirt, H. and Heberle-Bors, E. 1995. The D-type alfalfacyclin gene cycMs4 complements G1 cyclin-deficient yeast and is inducedin the G-1 phase of the cell cycle. Plant Cell 7(11):1847-1857.

[0145] Murray, J. A. H., Freeman, D., Greenwood, J., Huntley, R.,Makkerk, J. Riou-Khamlichi, C., Sorrell, D. A., Cockcroft, C.,Carmichael., J. P., Soni, R. and Shah, Z. H. 1998. Plant D cyclins andretinoblastoma protein homologues. In: Plant Cell Division, (Francis,D., Dudits, D. and Inze D., eds.), Portland Press, London.

[0146] Fuerst, R. A. U. A., Soni, R., Murray, J. A. H. and Lindsey, K.1998. Modulation of cyclin transcript levels in cultured cells ofArabidopsis thaliana. Plant Physiol. 112:1023-1033.

[0147] Hadwiger, J. A., Wittenberg, C., Richardson, H. E., de BarrosLopes, M. and Reed, S. I. 1989. A family of cyclin homologs that controlthe G1 phase in yeast. Proc. Natl. Acad. Sci. USA 86(16):6255-6259.

[0148] Matsushime, H., Roussel, M. F. and Sherr, C. J. 1991. Novelmammalian cyclins (CYL genes) expressed during G1. Cold Spring Harb.Symp. Quant. Biol. 56:69-74.

[0149] Richardson, H. E., Wittenberg, C., Cross, F and Reed, S. I. 1989.An essential G1 function for cyclin-like proteins in yeast. Cell59(6):1127-1133.

[0150] Soni, R., Carmichael, J. P., shah, Z. H. and Murray, J. A. H.1995. A family of cyclin D homologs from plants differentiallycontrolled by growth regulators and containing the conservedretinoblastoma protein interaction motif. Plant Cell 7:85-103.

[0151] The present invention provides, inter alia, compositions andmethods for modulating (i.e., increasing or decreasing) the total levelsof proteins of the present invention and/or altering their ratios inplants. Thus, the present invention provides utility in such exemplaryapplications as the regulation of cell division. The polypeptides of thepresent invention can be expressed at times or in quantities that arenot characteristic of non-recombinant plants.

[0152] In particular, modulating cell cycle proteins is expected toprovide a positive growth advantage and increase crop yield. Cell cyclenucleic acids can be adducted to a second nucleic acid sequence encodinga DNA-binding domain, for use in two-hybrid systems to identifyCycD-interacting proteins. It is expected that modulating the level ofcell cycle protein, i.e. over-expression, will increaseendoreduplication which is expected to increase the size of the seed,the size of the endosperm and amount of protein in the seed. The cellcycle protein can be used to affinity purify active maturation promotingfactor (MPF) or its components.

[0153] The present invention also provides isolated nucleic acidcomprising polynucleotides of sufficient length and complementarity to acell cycle gene to use as probes or amplification primers in thedetection, quantitation, or isolation of gene transcripts. For example,isolated nucleic acids of the present invention can be used as probes indetecting deficiencies in the level of mRNA in screenings for desiredtransgenic plants, for detecting mutations in the gene (e.g.,substitutions, deletions, or additions), for monitoring upregulation ofexpression or changes in enzyme activity in screening assays ofcompounds, for detection of any number of allelic variants(polymorphisms) of the gene, or for use as molecular markers in plantbreeding programs. The isolated nucleic acids of the present inventioncan also be used for recombinant expression of cell cycle polypeptides,or for use as immunogens in the preparation and/or screening ofantibodies. The isolated nucleic acids of the present invention can alsobe employed for use in sense or antisense suppression of one or morecell cycle genes in a host cell, tissue, or plant. Attachment ofchemical agents that bind, intercalate, cleave and/or crosslink to theisolated nucleic acids of the present invention can also be used tomodulate transcription or translation. Further, using a primer specificto an insertion sequence (e.g., transposon) and a primer whichspecifically hybridizes to an isolated nucleic acid of the presentinvention, one can use nucleic acid amplification to identity insertionsequence inactivated cell cycle genes from a cDNA library prepared frominsertion sequence mutagenized plants. Progeny seed from the plantscomprising the desired inactivated gene can be grown to a plant to studythe phenotypic changes characteristic of that inactivation. See, Toolsto Determine the Function of Genes, 1995 Proceedings of the FiftiethAnnual Corn and Sorghum Industry Research Conference, American SeedTrade Association, Washington, D.C., 1995. Additionally, non-translated5′ or 3′ regions of the polynucleotides of the present invention can beused to modulate turnover of heterologous mRNAs and/or proteinsynthesis. Further, the codon preference characteristic of thepolynucleotides of the present invention can be employed in heterologoussequences, or altered in homologous or heterologous sequences, tomodulate translational level and/or rates.

[0154] The present invention also provides isolated proteins comprisingpolypeptides including an amino acid sequence from the cell cyclepolypeptides (e.g., preproenzyme, proenzyme, or enzymes) as disclosedherein. The present invention also provides proteins comprising at leastone epitope from a cell cycle polypeptide. The proteins of the presentinvention can be employed in assays for enzyme agonists or antagonistsof enzyme function, or for use as immunogens or antigens to obtainantibodies specifically immunoreactive with a protein of the presentinvention. Such antibodies can be used in assays for expression levels,for identifying and/or isolating nucleic acids of the present inventionfrom expression libraries, or for purification of cell cyclepolypeptides.

[0155] The isolated nucleic acids of the present invention can be usedover a broad range of plant types, including species from the generaCucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis,Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus,Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura,Hyoscyamus, Lycopersicon, Nicotiana, Solanum, Petunia, Digitalis,Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus,Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum,Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum,Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum,Sorghum, Picea, and Populus. Preferred plants include corn, soybeans,sorghum, sunflower, wheat, rice, alfalfa and canola.

[0156] Nucleic Acids

[0157] The present invention provides, inter alia, isolated nucleicacids of RNA, DNA, and analogs and/or chimeras thereof, comprising acell cycle polynucleotide.

[0158] A. Polynucleotides Encoding A Protein of SEQ ID NOS: 2, 12, 14,or 22 or Conservatively Modified or Polymorphic Variants Thereof

[0159] The present invention provides isolated heterologous nucleicacids comprising a cell cycle polynucleotide, wherein the polynucleotideencodes a cell cycle polypeptide, disclosed herein in SEQ ID NOS: 2, 12,14, or 22, or conservatively modified or polymorphic variants thereof.Those of skill in the art will recognize that the degeneracy of thegenetic code allows for a plurality of polynucleotides to encode for theidentical amino acid sequence. Such “silent variations” can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Accordingly, the presentinvention includes polynucleotides of SEQ ID NOS: 1, 11, 13, or 21, andsilent variations of polynucleotides encoding a polypeptide of SEQ IDNOS: 2, 12, 14, or 22. The present invention further provides isolatednucleic acids comprising polynucleotides encoding conservativelymodified variants of a polypeptide of SEQ ID NOS: 2, 12, 14, or 22.Conservatively modified variants can be used to generate or selectantibodies immunoreactive to the non-variant polypeptide.

[0160] B. Polynucleotides Amplified from a Zea mays Nucleic Acid Library

[0161] As indicated in (b), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotides are amplified from a Zea mays nucleic acid library.Zea mays lines B73, PHRE1, A632, BMS-P2#10, W23, and Mol7 are known andpublicly available. Other publicly known and available maize lines canbe obtained from the Maize Genetics Cooperation (Urbana, Ill.).

[0162] The nucleic acid library may be a cDNA library, a genomiclibrary, or a library generally constructed from nuclear transcripts atany stage of intron processing. Generally, a cDNA nucleic acid librarywill be constructed to comprise a majority of full-length cDNAs. Often,cDNA libraries will be normalized to increase the representation ofrelatively rare cDNAs.

[0163] Total RNA Isolation: Libraries can be made from a variety ofmaize tissues but for optimal results one should isolate RNA's frommitotically active tissues such as shoot meristems, shoot meristemcultures, callus and suspension cultures, immature ears and tassels, andyoung seedlings. Since cell cycle proteins are typically expressed atspecific cell cycle stages it may be possible to enrich for such raremessages using exemplary cell cycle inhibitors such as aphidicolin,hydroxyurea, mimosine, and double-phosphate starvation methods to blockcells at the G1/S boundary. Cells can also be blocked at this stageusing the double phosphate starvation method. Hormone treatments thatstimulate cell division, for example cytokinin, would also increaseexpression of the cell cycle RNA.

[0164] Full length cDNA libraries from such rapidly-dividing tissues (orcells at the G1/S boundary) would provide opportunities for identifyingfull length, cell cycle related cDNAs. Full length cDNA libraries can beconstructed using the “Biotinylated CAP Trapper” method (Carninci, P.,et al., Genomics 37:327-336, 1996) or the “mRNA Cap Retention Procedure”(Edery, I., et al., Molecular and Cellular Biology 15:3363-3371, 1995).Full length cDNA libraries can be normalized to provide a higherprobability of sampling genes that express at low levels. Examples ofcDNA library normalization methods are summarized by Bento Soares(Bonaldo, M. F., et al., Genome Research 6:791-806, 1996).

[0165] Functional fragments of cell cycle protein can be identifiedusing a variety of techniques such as restriction analysis, Southernanalysis, primer extension analysis, and DNA sequence analysis. Functioncan also be determined by complementing yeast strains known to be mutantfor G1 cell cycle proteins with maize homologs. Primer extensionanalysis or S1 nuclease protection analysis, for example, can be used tolocalize the putative start site of transcription of the cloned gene.Ausubel at pages 4.8.1 to 4.8.5; Walmsley et al., “Quantitative andQualitative Analysis of Exogenous Gene Expression by the S1 NucleaseProtection Assay,” in Methods in Molecular Biology, Vol. 7, GeneTransfer and Expression.

[0166] The general approach of such functional analysis involvessubcloning DNA fragments of a genomic clone, cDNA clone or synthesizedgene sequence into an expression vector, introducing the expressionvector into a heterologous host, and relying on an assay system such asBrdU incorporation to monitor DNA synthesis in conjunction with variouswell-established visual methods to follow cell division (e.g. see T.Motomura, Cell cycle analysis in a multinucleate green alga, Boergensiaforbesti (Syphonoclades, Chlorophyta). Phycological Res. 44(1): 11-17,and J. L. Kennard et al., Pre-mitotic nuclear migration in subsidiarymother cells of Tradescantia occurs in the G1 of the cell cycle. CellMotility and the Cytoskeleton 36:55-67). Methods for generatingfragments of a cDNA or genomic clone are well known. In addition,variants can be obtained, for example, by oligonucleotide-directedmutagenesis, linker-scanning mutagenesis, mutagenesis using thepolymerase chain reaction, and the like. See, for example, Ausubel,pages 8.0.3-8.5.9. Also, see generally, McPherson (ed.), DirectedMutagenesis: A Practical Approach, (IRL Press, 1991). Thus, the presentinvention also encompasses DNA molecules comprising nucleotide sequencesthat have substantial sequence similarity with SEQ ID NO: 1, 11, 13, or22 and encode CycD.

[0167] The polynucleotides of the present invention include thoseamplified using the following primer pairs:

[0168] Primer Sets for ZmCycDa-1 Primer sets for ZmCycDa-1 1) Primersets flanking ZmCycDa-1 cDNA: Set #1: For015′ GCAAGCATGGTGCCGGGCTATGACTGC 3′ Rev015′ AGCGGTGAGGAGCACACCTGAAGCGTACCA 3′ Set #2: For015′ GCAAGCATGGTGCCGGGCTATGACTGC 3′ Rev02 5′ TCTATTCCTCTGCCGACCCCCATCCTT3′ Set #3: For02 5′ CCCCTCTCCACTTGAGAAGAACACAATTAG 3′ Rev015′ AGCGGTGAGGAGCACACCTGAAGCGTACCA 3′ Set #4: For025′ CCCCTCTCCACTTGAGAAGAACACAATTAG 3′ Rev025′ TCTATTCCTCTGCCGACCCCCATCCTT 3′ 2) Primer sets inside ZmCycDa-1 cDNA:Set #1: For01 5′ CGGGCTATGACTGCGCCGCCTCCGT 3′ Rev015′ CTCCTCTTGCTTGTGGAAGAACTATGG 3′ Set #2: For02:5′ ATGGTGCCGGGCTATGACTGCGCCG 3′ Rev02: 5′ TTAGAGTAGACGTCTAGTGATCCTT 3′Primer sets for ZmCycDb-1: 1) Primer sets flanking ZmCycDb-1: Set #1For01: 5′ CAGACTTTGACTTGCTGGTGTCCGGT 3′ Rev01:5′ GCCGCCTCTCAATGCACTCTTTG 3′ Set #2 For02: 5′ TGGGAGTGAGATACGCCGGTACAGA3′ Rev02: 5′ TCCCATCGGATCTCCTCTAGCGCCC 3′ 2) Primer sets insideZmyCycDb-1: For01: 5′ CACGCGCACCAGCCCACCGCCCAG 3′ Rev01:5′ TCCCATCGGATCTCCTCTAGCGCCC 3′ Set #2 For02: 5′ TCACTCTTTGGTCCATTGGGC3′ Rev02: 5′ ATGGCGCCGAGCTGCTACGA 3′ Primer sets for ZmCycDc-1: 1)Primer sets flanking ZmCycDc-1 cDNA: Set #1: For01:CAGTACCCCCACGCTGCACAG Rev01: TCACGCTTGTTCTGTCGTCTTTACAC Set #2: For02:GCTGCTGCAAGTCCGCAACCACTG Rev02: CGCTTGTTCTGTCGTCTTTACACTG 2) Primer setsinside ZmCycDc-1 cDNA: Set #1: For01: 5′ ACCTCCATCCTCATCTGCCTGGAAGACRev01: 5′ CTGGACTGCACTGCACTGCAATGC Set #2: For02:5′ CATCCTCATCTGCCTGGAAGACGGC Rev02: 5′ AATGCACTGCCAGCAGCTGAGCT

[0169] The present invention also provides subsequences of full-lengthnucleic acids. Any number of subsequences can be obtained by referenceto SEQ ID NOS: 1, 11, 13, or 21, and using primers which selectivelyamplify, under stringent conditions to: at least two sites to thepolynucleotides of the present invention, or to two sites within thenucleic acid which flank and comprise a polynucleotide of the presentinvention, or to a site within a polynucleotide of the present inventionand a site within the nucleic acid which comprises it. A variety ofmethods for obtaining 5′ and/or 3′ ends is well known in the art. See,e.g., RACE (Rapid Amplification of Complementary Ends) as described inFrohman, M. A., in PCR Protocols: A Guide to Methods and Applications,M. A. Innis, D. H. Gelfand, J. J. Sninsky, T. J. White, Eds. (AcademicPress, Inc., San Diego, 1990), pp. 28-38.); see also, U.S. Pat. No.5,470,722, and Current Protocols in Molecular Biology, Unit 15.6,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995). Thus, the present invention provides cell cyclepolynucleotides having the sequence of the cell cycle gene, nucleartranscript, cDNA, or complementary sequences and/or subsequencesthereof.

[0170] Primer sequences can be obtained by reference to a contiguoussubsequence of a polynucleotide of the present invention. Primers arechosen to selectively hybridize, under PCR amplification conditions, toa polynucleotide of the present invention in an amplification mixturecomprising a genomic and/or cDNA library from the same species.Generally, the primers are complementary to a subsequence of theamplicon they yield. In some embodiments, the primers will beconstructed to anneal at their 5′ terminal end's to the codon encodingthe carboxy or amino terminal amino acid residue (or the complementsthereof of the polynucleotides of the present invention. The primerlength in nucleotides is selected from the group of integers consistingof from at least 15 to 50. Thus, the primers can be at least 15, 18, 20,25, 30, 40, or 50 nucleotides in length. A non-annealing sequence at the5′ end of the primer (a “tail”) can be added, for example, to introducea cloning site at the terminal ends of the amplicon.

[0171] The amplification primers may optionally be elongated in the 3′direction with additional contiguous nucleotides from the polynucleotidesequences, such as SEQ ID NOS: 1, 11, 13, or 21, from which they arederived. The number of nucleotides by which the primers can be elongatedis selected from the group of integers consisting of from at least 1 to25. Thus, for example, the primers can be elongated with an additional1, 5, 10, or 15 nucleotides. Those of skill will recognize that alengthened primer sequence can be employed to increase specificity ofbinding (i.e., annealing) to a target sequence.

[0172] The amplification products can be translated using expressionsystems well known to those of skill in the art and as discussed, infra.The resulting translation products can be confirmed as polypeptides ofthe present invention by, for example, assaying for the appropriatecatalytic activity (e.g., specific activity and/or substratespecificity), or verifying the presence of one or more linear epitopesthat are specific to a polypeptide of the present invention. Methods forprotein synthesis from PCR derived templates are known in the art andavailable commercially. See, e.g., Amersham Life Sciences, Inc., Catalog'97, p.354.

[0173] C. Polynucleotides Which Selectively Hybridize to aPolynucleotide of (A) or (B)

[0174] As indicated in (c), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotides selectively hybridize, under selective hybridizationconditions, to a polynucleotide of paragraphs (A) or (B) as discussed,supra. Thus, the polynucleotides of this embodiment can be used forisolating, detecting, and/or quantifying nucleic acids comprising thepolynucleotides of (A) or (B). For example, polynucleotides of thepresent invention can be used to identify, isolate, or amplify partialor full-length clones in a deposited library. In some embodiments, thepolynucleotides are genomic or cDNA sequences isolated from a Zea maysnucleic acid library. Preferably, the cDNA library comprises at least80% full-length sequences, preferably at least 85% or 90% full-lengthsequences, and more preferably at least 95% full-length sequences. ThecDNA libraries can be normalized to increase the representation of raresequences. Low stringency hybridization conditions are typically, butnot exclusively, employed with sequences having a reduced sequenceidentity relative to complementary sequences. Moderate and highstringency conditions can optionally be employed for sequences ofgreater identity. Low stringency conditions allow selectivehybridization of sequences having about 70% sequence identity and can beemployed to identify orthologous or paralogous sequences.

[0175] D. Polynucleotides Having at Least 60% Sequence Identity with thePolynucleotides of (A), (B) or (C)

[0176] As indicated in (d), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotides have a specified identity at the nucleotide level toa polynucleotide as disclosed above in paragraphs (A), (B), or (C). Thepercentage of identity to a reference sequence is at least 60% and,rounded upwards to the nearest integer, can be expressed as an integerselected from the group of integers consisting of from 60 to 99. Thus,for example, the percentage of identity to a reference sequence can beat least 70%, 75%, 80%, 85%, 90%, or 95%.

[0177] Optionally, the polynucleotides of this embodiment will share anepitope with a polypeptide encoded by the polynucleotides of (A), (B),or (C). Thus, these polynucleotides encode a first polypeptide thatelicits production of antisera comprising antibodies that arespecifically reactive to a second polypeptide encoded by apolynucleotide of (A), (B), or (C). However, the first polypeptide doesnot bind to antisera raised against itself when the antisera has beenfully immunosorbed with the first polypeptide. Hence, thepolynucleotides of this embodiment can be used to generate antibodiesfor use in, for example, the screening of expression libraries fornucleic acids comprising polynucleotides of (A), (B), or (C), or forpurification of, or in immunoassays for, polypeptides encoded by thepolynucleotides of (A), (B), or (C). The polynucleotides of thisembodiment embrace nucleic acid sequences that can be employed forselective hybridization to a polynucleotide encoding a polypeptide ofthe present invention.

[0178] Screening polypeptides for specific binding to antisera can beconveniently achieved using peptide display libraries. This methodinvolves the screening of large collections of peptides for individualmembers having the desired function or structure. Antibody screening ofpeptide display libraries is well known in the art. The displayedpeptide sequences can be from 3 to 5000 or more amino acids in length,frequently from 5-100 amino acids long, and often from about 8 to 15amino acids long. In addition to direct chemical synthetic methods forgenerating peptide libraries, several recombinant DNA methods have beendescribed. One type involves the display of a peptide sequence on thesurface of a bacteriophage or cell. Each bacteriophage or cell containsthe nucleotide sequence encoding the particular displayed peptidesequence. Such methods are described in PCT patent publication Nos.91/17271, 91/18980, 91/19818, and 93/08278. Other systems for generatinglibraries of peptides have aspects of both in vitro chemical synthesisand recombinant methods. See, PCT Patent publication Nos. 92/05258,92/14843, and 96/19256. See also, U.S. Pat. Nos. 5,658,754; and5,643,768. Peptide display libraries, vectors, and screening kits arecommercially available from such suppliers as Invitrogen (Carlsbad,Calif.).

[0179] E. Polynucleotides Encoding a Protein Having a Subsequence from aPrototype Polypeptide and is Cross-Reactive to the Prototype Polypeptide

[0180] As indicated in (e), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotides encode a protein having a subsequence of contiguousamino acids from a prototype cell cycle polypeptide. Exemplary prototypecell cycle polypeptides are provided in SEQ ID NOS. 2, 12, 14, or 23.The length of contiguous amino acids from the prototype polypeptide isselected from the group of integers consisting of from at least 10 tothe number of amino acids within the prototype sequence. Thus, forexample, the polynucleotide can encode a polypeptide having asubsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50,contiguous amino acids from the prototype polypeptide. Further, thenumber of such subsequences encoded by a polynucleotide of the instantembodiment can be any integer selected from the group consisting of from1 to 20, such as 2, 3, 4, or 5. The subsequences can be separated by anyinteger of nucleotides from 1 to the number of nucleotides in thesequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides.

[0181] The proteins encoded by polynucleotides of this embodiment, whenpresented as an immunogen, elicit the production of polyclonalantibodies which specifically bind to a prototype polypeptide such as,but not limited to, a polypeptide encoded by the polynucleotide of (b),supra, or exemplary polypeptides of SEQ ID NOS. 2, 12, 14, or 23.Generally, however, a protein encoded by a polynucleotide of thisembodiment does not bind to antisera raised against the prototypepolypeptide when the antisera have been fully immunosorbed with theprototype polypeptide. Methods of making and assaying for antibodybinding specificity/affinity are well known in the art. Exemplaryimmunoassay formats include ELISA, competitive immunoassays,radioimmunoassays, Western blots, indirect immunofluorescent assays andthe like.

[0182] In a preferred assay method, fully immunosorbed and pooledantisera that is elicited to the prototype polypeptide can be used in acompetitive binding assay to test the protein. The concentration of theprototype polypeptide required to inhibit 50% of the binding of theantisera to the prototype polypeptide is determined. If the amount ofthe protein required to inhibit binding is less than twice the amount ofthe prototype protein, then the protein is said to specifically bind tothe antisera elicited to the immunogen. Accordingly, the proteins of thepresent invention embrace allelic variants, conservatively modifiedvariants, and minor recombinant modifications to a prototypepolypeptide.

[0183] A polynucleotide of the present invention optionally encodes aprotein having a molecular weight as the non-glycosylated protein within20% of the molecular weight of the full-length non-glycosylated cellcycle polypeptides as disclosed herein. Molecular weight can be readilydetermined by SDS-PAGE under reducing conditions. Preferably, themolecular weight is within 15% of a full-length cell cycle polypeptide,more preferably within 10% or 5%, and most preferably within 3%, 2%, or1% of a full-length cell cycle polypeptide of the present invention.Molecular weight determination of a protein can be convenientlyperformed by SDS-PAGE under denaturing conditions.

[0184] Optionally, the polynucleotides of this embodiment will encode aprotein having a specific activity at least 20%, 30%, 40%, or 50% of thenative, endogenous (i.e., non-isolated), full-length cell cyclepolypeptide. Further, the proteins encoded by polynucleotides of thisembodiment will optionally have a substantially similar apparentdissociation constant (K_(m)) and/or catalytic activity (i.e., themicroscopic rate constant, k_(cat)) as the native endogenous,full-length cell cycle protein. Those of skill in the art will recognizethat k_(cat)/K_(m) value determines the specificity for competingsubstrates and is often referred to as the specificity constant.Proteins of this embodiment can have a k_(cat)/K_(m) value at least 10%of the non-isolated full-length cell cycle polypeptide as determinedusing the substrate of that polypeptide from the cell cycle specificpathways, supra. Optionally, the k_(cat)/K_(m) value will be at least20%, 30%, 40%, 50%, and most preferably at least 60%, 70%, 80%, 90%, or95% the k_(cat)/K_(m) value of the non-isolated, full-length cell cyclepolypeptide. Determination of k_(cat), K_(m), and k_(cat)/K_(m) can bedetermined by any number of means well known to those of skill in theart. For example, the initial rates (i.e., the first 5% or less of thereaction) can be determined using rapid mixing and sampling techniques(e.g., continuous-flow, stopped-flow, or rapid quenching techniques),flash photolysis, or relaxation methods (e.g., temperature jumps) inconjunction with such exemplary methods of measuring asspectrophotometry, spectrofluorimetry, nuclear magnetic resonance, orradioactive procedures. Kinetic values are conveniently obtained using aLineweaver-Burk or Eadie-Hofstee plot.

[0185] F. Polynucleotides Complementary to the Polynucleotides of(A)-(E)

[0186] As indicated in (f), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotides are complementary to the polynucleotides ofparagraphs A-E, above. As those of skill in the art will recognize,complementary sequences base-pair throughout the entirety of theirlength with the polynucleotides of (A)-(E) (i.e., have 100% sequenceidentity over their entire length). Complementary bases associatethrough hydrogen bonding in double stranded nucleic acids. For example,the following base pairs are complementary: guanine and cytosine;adenine and thymine; and adenine and uracil.

[0187] G. Polynucleotides that are Subsequences of the Polynucleotidesof (A)-(F)

[0188] As indicated in (g), supra, the present invention providesisolated nucleic acids comprising cell cycle polynucleotides, whereinthe polynucleotide comprises at least 15 contiguous bases from thepolynucleotides of (A) through (F) as discussed above. The length of thepolynucleotide is given as an integer selected from the group consistingof from at least 15 to the length of the nucleic acid sequence fromwhich the polynucleotide is a subsequence of. Thus, for example,polynucleotides of the present invention are inclusive ofpolynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or100 contiguous nucleotides in length from the polynucleotides of(A)-(F). Optionally, the number of such subsequences encoded by apolynucleotide of the instant embodiment can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4, or 5. Thesubsequences can be separated by any integer of nucleotides from 1 tothe number of nucleotides in the sequence such as at least 5, 10, 15,25, 50, 100, or 200 nucleotides.

[0189] The subsequences of the present invention can comprise structuralcharacteristics of the sequence from which it is derived. Alternatively,the subsequences can lack certain structural characteristics of thelarger sequence from which it is derived. For example, a subsequencefrom a polynucleotide encoding a polypeptide having at least one linearepitope in common with a prototype sequence, may encode an epitope incommon with the prototype sequence. Alternatively, the subsequence maynot encode an epitope in common with the prototype sequence but can beused to isolate the larger sequence by, for example, nucleic acidhybridization with the sequence from which it's derived. Subsequencescan be used to modulate or detect gene expression by introducing intothe subsequence compounds that bind, intercalate, cleave and/orcrosslink to nucleic acids. Exemplary compounds include acridine,psoralen, phenanthroline, naphthoquinone, daunomycin orchloroethylaminoaryl conjugates.

[0190] Construction of Nucleic Acids

[0191] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention will be cloned, amplified, or otherwise constructedfrom a monocot. In preferred embodiments the monocot is Zea mays.Particularly preferred is the use of Zea mays tissue from tassel andvegetative meristem.

[0192] The nucleic acids may conveniently comprise sequences in additionto a polynucleotide of the present invention. For example, amulti-cloning site comprising one or more endonuclease restriction sitesmay be inserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention. The nucleic acidof the present invention—excluding the polynucleotide sequence—isgenerally a vector, adapter, or linker for cloning and/or expression ofa polynucleotide of the present invention. Use of cloning vectors,expression vectors, adapters, and linkers is well known in the art.Exemplary nucleic acids include such vectors as: M13, lambda ZAPExpress, lambda ZAP II, lambda gt10, lambda gt11, pBK-CMV, pBK-RSV,pBluescript II, lambda DASH II, lambda EMBL 3, lambda EMBL4, pWE15,SuperCos 1, SurfZap, Uni-ZAP, pBC, pBS+/−, pSG5, pBK, pCR-Script, pET,pSPUTK, p3′SS, pOPRSVI CAT, pOPI3 CAT, pXT1, pSG5, pPbac, pMbac,pMC1neo, pOG44, pOG45, pFRTβGAL, pNEOβGAL, pRS403, pRS404, pRS405,pRS406, pRS413, pRS414, pRS415, pRS416, lambda MOSSlox, and lambdaMOSElox. For a description of various nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla,Calif.); and, Amersham Life Sciences, Inc., Catalog '97 (ArlingtonHeights, Ill.).

[0193] A. Recombinant Methods for Constructing Nucleic Acids

[0194] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probesthat selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library. While isolation ofRNA and construction of cDNA and genomic libraries is well known tothose of ordinary skill in the art, the following highlights some of themethods employed.

[0195] A1. mRNA Isolation and Purification

[0196] Total RNA from plant cells comprises such nucleic acids asmitochondrial RNA, chloroplastic RNA, rRNA, tRNA, hnRNA and mRNA. TotalRNA preparation typically involves lysis of cells and removal ofproteins, followed by precipitation of nucleic acids. Extraction oftotal RNA from plant cells can be accomplished by a variety of means.Frequently, extraction buffers include a strong detergent such as SDSand an organic denaturant such as guanidinium isothiocyanate, guanidinehydrochloride or phenol. Following total RNA isolation, poly(A)⁺ mRNA istypically purified from the remainder RNA using oligo(dT) cellulose.Exemplary total RNA and mRNA isolation protocols are described in PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel, etal., Eds., Greene Publishing and Wiley-Interscience, New York (1995).Total RNA and mRNA isolation kits are commercially available fromvendors such as Stratagene (La Jolla, Calif.), Clonetech (Palo Alto,Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli, Pa.). See also,U.S. Pat. Nos. 5,614,391; and, 5,459,253. The mRNA can be fractionatedinto populations with size ranges of about 0.5, 1.0, 1.5, 2.0, 2.5 or3.0 kb. The cDNA synthesized for each of these fractions can be sizeselected to the same size range as its mRNA prior to vector insertion.This method helps eliminate truncated cDNA formed by incompletelyreverse transcribed mRNA.

[0197] A2. Construction of a cDNA Library

[0198] Construction of a cDNA library generally entails five steps.First, first strand cDNA synthesis is initiated from a poly(A)⁺ mRNAtemplate using a poly(dT) primer or random hexanucleotides. Second, theresultant RNA-DNA hybrid is converted into double stranded cDNA,typically by a combination of RNAse H and DNA polymerase I (or Klenowfragment). Third, the termini of the double stranded cDNA are ligated toadaptors. Ligation of the adaptors will produce cohesive ends forcloning. Fourth, size selection of the double stranded cDNA eliminatesexcess adaptors and primer fragments, and eliminates partial cDNAmolecules due to degradation of mRNAs or the failure of reversetranscriptase to synthesize complete first strands. Fifth, the cDNAs areligated into cloning vectors and packaged. cDNA synthesis protocols arewell known to the skilled artisan and are described in such standardreferences as: Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997); and, Current Protocols in MolecularBiology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995). cDNA synthesis kits are availablefrom a variety of commercial vendors such as: Stratagene, and Pharmacia.

[0199] A number of cDNA synthesis protocols have been described whichprovide substantially pure full-length cDNA libraries. Substantiallypure full-length cDNA libraries are constructed to comprise at least90%, and more preferably at least 93% or 95% full-length inserts amongstclones containing inserts. The length of insert in such libraries can befrom 0 to 8, 9, 10, 11, 12, 13, or more kilobase pairs. Vectors toaccommodate inserts of these sizes are known in the art and availablecommercially. See, e.g., the Stratagene lambda ZAP Express (cDNA cloningvector with 0 to 12 kb cloning capacity).

[0200] An exemplary method of constructing a greater than 95% purefull-length cDNA library is described by Carninci et al., Genomics37:327-336 (1996). In that protocol, the cap-structure of eukaryoticmRNA is chemically labeled with biotin. By using streptavidin-coatedmagnetic beads, only the full-length first-strand cDNA/mRNA hybrids areselectively recovered after RNase I treatment. The method provides ahigh yield library with an unbiased representation of the starting mRNApopulation. Other methods for producing full-length libraries are knownin the art. See, e.g., Edery et al., Mol. Cell Biol.15(6):3363-3371(1995); and, PCT Application WO 96/34981.

[0201] A3. Normalized or Subtracted cDNA Libraries

[0202] A non-normalized cDNA library represents the mRNA population ofthe tissue it was made from. Since unique clones are out-numbered byclones derived from highly expressed genes their isolation can belaborious. Normalization of a cDNA library is the process of creating alibrary in which each clone is more equally represented.

[0203] A number of approaches to normalize cDNA libraries are known inthe art. One approach is based on hybridization to genomic DNA. Thefrequency of each hybridized cDNA in the resulting normalized librarywould be proportional to that of each corresponding gene in the genomicDNA. Another approach is based on kinetics. If cDNA reannealing followssecond-order kinetics, rarer species anneal less rapidly and theremaining single-stranded fraction of cDNA becomes progressively morenormalized during the course of the hybridization. Specific loss of anyspecies of cDNA, regardless of its abundance, does not occur at any Cotvalue. Construction of normalized libraries is described in Ko, Nucl.Acids. Res., 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl.Acad. U.S.A. 88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685, and5,637,685. In an exemplary method described by Soares et al.,normalization resulted in reduction of the abundance of clones from arange of four orders of magnitude to a narrow range of only 1 order ofmagnitude, Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

[0204] Subtracted cDNA libraries are another means to increase theproportion of less abundant cDNA species. In this procedure, cDNAprepared from one pool of mRNA is depleted of sequences present in asecond pool of mRNA by hybridization. The cDNA:mRNA hybrids are removedand the remaining un-hybridized cDNA pool is enriched for sequencesunique to that pool. See, Foote et al. in, Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997); Kho andZarbl, Technique, 3(2):58-63 (1991); Sive and St. John, Nucl. AcidsRes., 16(22):10937 (1988); Current Protocols in Molecular Biology,Ausubel, et al., Eds., Greene Publishing and Wiley-Interscience, NewYork (1995); and, Swaroop et al., Nucl. Acids Res., 19(8):1954 (1991).cDNA subtraction kits are commercially available. See, e.g., PCR-Select(Clontech).

[0205] A4. Construction of a Genomic Library

[0206] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation, e.g. using restriction endonucleases,and are ligated with vector DNA to form concatemers that can be packagedinto the appropriate vector. Methodologies to accomplish these ends, andsequencing methods to verify the sequence of nucleic acids are wellknown in the art. Examples of appropriate molecular biologicaltechniques and instructions sufficient to direct persons of skillthrough many construction, cloning, and screening methodologies arefound in Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Vols. 1-3 (1989), Methods inEnzymology, Vol. 152, Guide to Molecular Cloning Techniques, Berger andKimmel, Eds., San Diego: Academic Press, Inc. (1987), Current Protocolsin Molecular Biology, Ausubel, et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); Plant Molecular Biology: ALaboratory Manual, Clark, Ed., Springer-Verlag, Berlin (1997). Kits forconstruction of genomic libraries are also commercially available.

[0207] A5. Nucleic Acid Screening and Isolation Methods

[0208] The cDNA or genomic library can be screened using a probe basedupon the sequence of a polynucleotide of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous genes in the same or differentplant species. Those of skill in the art will appreciate that variousdegrees of stringency of hybridization can be employed in the assay; andeither the hybridization or the wash medium can be stringent. As theconditions for hybridization become more stringent, there must be agreater degree of complementarity between the probe and the target forduplex formation to occur. The degree of stringency can be controlled bytemperature, ionic strength, pH and the presence of a partiallydenaturing solvent such as formamide. For example, the stringency ofhybridization is conveniently varied by changing the polarity of thereactant solution through manipulation of the concentration of formamidewithin the range of 0% to 50%. The degree of complementarity (sequenceidentity) required for detectable binding will vary in accordance withthe stringency of the hybridization medium and/or wash medium. Thedegree of complementarity will optimally be 100 percent; however, itshould be understood that minor sequence variations in the probes andprimers may be compensated for by reducing the stringency of thehybridization and/or wash medium.

[0209] The nucleic acids of interest can also be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related genes directly fromgenomic DNA or cDNA libraries. PCR and other in vitro amplificationmethods may also be useful, for example, to clone nucleic acid sequencesthat code for proteins to be expressed, to clone flanking genomicsequences, 5′ untranslated regions and 3′ sequences, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes. Examples oftechniques sufficient to direct persons of skill through in vitroamplification methods are found in Berger, Sambrook, and Ausubel, aswell as Mullis et al., U.S. Pat. No. 4,683,202 (1987); and, PCRProtocols A Guide to Methods and Applications, Innis et al., Eds.,Academic Press Inc., San Diego, Calif. (1990). Commercially availablekits for genomic PCR amplification are known in the art. See, e.g.,Advantage-GC Genomic PCR-Kit (Clontech). The T4 gene 32 protein(Boehringer Mannheim) can be used to improve yield of long PCR products.

[0210] PCR-based screening methods have also been described. Wilfingeret al. describe a PCR-based method in which the longest cDNA isidentified in the first step so that incomplete clones can be eliminatedfrom study. BioTechniques, 22(3): 481-486 (1997). In that method, aprimer pair is synthesized with one primer annealing to the 5′ end ofthe sense strand of the desired cDNA and the other primer to the vector.Clones are pooled to allow large-scale screening. By this procedure, thelongest possible clone is identified amongst candidate clones. Further,the PCR product is used solely as a diagnostic for the presence of thedesired cDNA and does not utilize the PCR product itself. Such methodsare particularly effective in combination with a full-length cDNAconstruction methodology, supra.

[0211] B. Synthetic Methods for Constructing Nucleic Acids

[0212] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99 (1979);the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triestermethod described by Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res.,12:6159-6168 (1984); and, the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis generally produces a single strandedoligonucleotide. This may be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. One of skill willrecognize that while chemical synthesis of DNA is limited to sequencesof about 100 bases, longer sequences may be obtained by the ligation ofshorter sequences.

[0213] Recombinant Expression Cassettes

[0214] The present invention further provides recombinant expressioncassettes comprising a nucleic acid of the present invention. A nucleicacid sequence coding for the desired polynucleotide of the presentinvention, for example a cDNA or a genomic sequence encoding a fulllength polypeptide of the present invention, can be used to construct arecombinant expression cassette which can be introduced into the desiredhost cell. A recombinant expression cassette will typically comprise apolynucleotide of the present invention operably linked totranscriptional initiation regulatory sequences which will direct thetranscription of the polynucleotide in the intended host cell, such astissues of a transformed plant.

[0215] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible or constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0216] Cell cycle vectors were constructed using standard molecularbiology techniques. See, for example, Sambrook et al. (eds.) MolecularCloning: a Laboratory Manual, Second Edition, (Cold Spring HarborLaboratory Press, cold Spring Harbor, N.Y. 1989). Plasmids are based onpUC18. The vectors used in these experiments contain combinations of thesame basic regulatory elements. The Omega prime (O′) 5-prine sequence isdescribed by Gallie et al., Nucl. Acids Res. 15:3257-3273 (1987). Theselective marker gene, bar (Thompson et al., EMBO J. 6:2519-2523(1987)), was used in conjunction with bialaphos selection to recovertransformants. The Cauliflower Mosaic Virus 35S promoter with aduplicated enhancer region is described by Gardner et al., Nucl. AcidRes. 9:2871-2888 (1981). The 79 bp Tobacco Mosaic Virus leader isdescribed by Gallie et al., Nucl. Acid Res. 15:3257-3273 (1987) and wasinserted downstream of the promoter followed by the first intron of themaize alcohol dehydrogenase gene ADH1-S. Described by Dennis et al.,Nucl. Acid Res. 12:3983-3990 (1984). The 3′ sequence pinII is describedby An et al., Plant Cell 1:115-122 (1989).

[0217] A plant promoter fragment can be employed which will directexpression of a polynucleotide of the present invention in all tissuesof a regenerated plant. Such promoters are referred to herein as“constitutive” promoters and are active under most environmentalconditions and states of development or cell differentiation. Examplesof constitutive promoters include the cauliflower mosaic virus (CaMV)35S transcription initiation region, the 1′- or 2′-promoter derived fromT-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smaspromoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No.5,683,439), the Nos promoter, the pEmu promoter, the rubisco promoter,the GRP1-8 promoter, and other transcription initiation regions fromvarious plant genes known to those of skill.

[0218] Promoters

[0219] A. Inducible Promoters

[0220] An inducible promoter can be operably linked to a nucleotidesequence encoding ZmCycD. Optionally, the inducible promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a nucleotide sequence encoding ZmCycD. With aninducible promoter the rate of transcription increases in response to aninducing agent.

[0221] Any inducible promoter can be used in the instant invention. SeeWard et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary induciblepromoters include that from the ACE1 system which responds to copper(Mett et al., PNAS 90:4567-4571 (1993)); In2 gene from maize whichresponds to benzenesulfonamide herbicide safeners (Hershey et al., Mol.Gen. Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genet. 227:229-237 (1991). A particularly preferred inducible promoteris a promoter that responds to an inducing agent to which plants do notnormally respond. An exemplary inducible promoter is the induciblepromoter from a steroid hormone gene the transcriptional activity ofwhich is induced by a glucocorticosteroid hormone. Schena et al., Proc.Natl. Acad. Sci. U.S.A. 88:10421 (1991).

[0222] The expression vector comprises an inducible promoter operablylinked to a nucleotide sequence encoding ZmCycD. The expression vectoris introduced into plant cells and presumptively transformed cells areexposed to an inducer of the inducible promoter. The cells can bescreened for the presence of ZmCycD protein by northern, RPA, or RT-PCR(using transgene specific probes/oligo pairs) BrdU or cell divisionassays, as described above.

[0223] B. Tissue-specific or Tissue Preferred Promoters

[0224] A tissue-specific promoter can be operably linked to a nucleotidesequence encoding a ZmCycD protein. Optionally, the tissue-specificpromoter is operably linked to a nucleotide sequence encoding a signalsequence which is operably linked to a nucleotide sequence encodingZmCycD. Plants transformed with a gene encoding ZmCycD operably linkedto a tissue-specific promoter produce the ZmCycD protein exclusively, orpreferentially, in a specific tissue.

[0225] Any tissue-specific or tissue-preferred promoter can be utilizedin the instant invention. Exemplary tissue-specific or tissue-preferredpromoters include a seed-preferred promoter such as that from thephaseolin gene (Murai et al., Science 23:476-482 (1983) andSengupta-Gopalan et al., Proc. Natl. Acad. Sci. USA 82:3320-3324(1985)), napin promoter, β-conglycinin promoter soybean lectin promoter,maize 15 kD zein promoter, 22 kD zein promoter, γ-zein promoter, waxypromoter, shrunken 1 promoter, globulin 1 promoter and shrunken 2promoter (Thompson, et al.; BioEssays; Vol. 10; p.108; (1989); aleaf-specific and light-induced promoter such as that from cab orrubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko etal., Nature 318:579-582 (1985)); an anther-specific promoter such asthat from LAT52 (Twell et al., Mol. Gen. Genet. 217:240-245 (1989)); apollen-specific promoter such as that from Zm13 (Guerrero et al., Mol.Gen. Genet. 224:161-168 (1993)) or a microspore-preferred promoter suchas that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).

[0226] The expression vector comprises a tissue-specific ortissue-preferred promoter operably linked to a nucleotide sequenceencoding cell cycle protein. The expression vector is introduced intoplant cells. The cells are screened for the presence of cell cycleprotein by either BrdU or cell division assays, as described above.

[0227] C. Constitutive Promoters

[0228] A constitutive promoter can be operably linked to a nucleotidesequence encoding a cell cycle protein or the constitutive promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a nucleotide sequence encoding cell cycleprotein.

[0229] Many different constitutive promoters can be utilized in theinstant invention. Exemplary constitutive promoters include thepromoters from plant viruses such as the 35S promoter from CaMV (Odellet al., Nature 313:810-812 (1985)), Commelina yellow mottled virus (R.Torbert et al., Plant Cell Rep. 17:284-287 (1988)) and the promotersfrom such genes as rice actin (McElroy et al., Plant Cell 2:163-171(1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632(1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU(Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten etal., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al.,Mol. Gen. Genet. 231:276-285 (1992) and Atanassova et al., Plant Journal2(3):291-300 (1992)).

[0230] The ALS promoter, a Xbal/Ncol fragment 5-prime to the Brassicanapus ALS3 structural gene (or a nucleotide sequence that hassubstantial sequence similarity to the Xbal/Ncol fragment), represents aparticularly useful constitutive promoter. Co-pending Pioneer Hi-BredInternational U.S. patent application Ser. No. 08/409,297.

[0231] The expression vector comprises a constitutive promoter operablylinked to a nucleotide sequence encoding cell cycle protein. Theexpression vector is introduced into plant cells and presumptivelytransformed CELLS are screened for the presence of cell cycle protein byeither BrdU or cell division assays, as described above.

[0232] Alternatively, the plant promoter can direct expression of apolynucleotide of the present invention in a specific tissue or may beotherwise under more precise environmental or developmental control.Such promoters are referred to here as “inducible” promoters.Environmental conditions that may effect transcription by induciblepromoters include pathogen attack, anaerobic conditions, or the presenceof light. Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, and the PPDK promoter which is inducible bylight.

[0233] Examples of promoters under developmental control includepromoters that initiate transcription only, or preferentially, incertain tissues, such as leaves, roots, fruit, seeds, or flowers. Theoperation of a promoter may also vary depending on its location in thegenome. Thus, an inducible promoter may become fully or partiallyconstitutive in certain locations.

[0234] Both heterologous and non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inrecombinant expression cassettes to drive expression of antisensenucleic acids to reduce, increase, or alter cell cycle content and/orcomposition in a desired tissue. Thus, in some embodiments, the nucleicacid construct will comprise a promoter functional in a plant cell, suchas in Zea mays, operably linked to a polynucleotide of the presentinvention. Promoters useful in these embodiments include the endogenouspromoters driving expression of a polypeptide of the present invention.

[0235] In some embodiments, isolated nucleic acids which serve aspromoter or enhancer elements can be introduced in the appropriateposition (generally upstream) of a non-heterologous form of apolynucleotide of the present invention so as to up or down regulateexpression of a polynucleotide of the present invention. For example,endogenous promoters can be altered in vivo by mutation, deletion,and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling etal., PCT/US93/03868), or isolated promoters can be introduced into aplant cell-in the proper orientation and distance from a cell cycle geneso as to control the expression of the gene. Gene expression can bemodulated under conditions suitable for plant growth so as to alter cellcycle content and/or composition. Thus, the present invention providescompositions, and methods for making, heterologous promoters and/orenhancers operably linked to a native, endogenous (i.e.,non-heterologous) form of a polynucleotide of the present invention.

[0236] Methods for identifying promoters with a particular expressionpattern, in terms of, e.g., tissue type, cell type, stage ofdevelopment, and/or environmental conditions, are well known in the art.See, e.g., The Maize Handbook, Chapters 114-115, Freeling and Walbot,Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3^(rd) edition,Chapter 6, Sprague and Dudley, Eds., American Society of Agronomy,Madison, Wis. (1988). A typical step in promoter isolation methods isidentification of gene products that are expressed with some degree ofspecificity in the target tissue. Amongst the range of methodologiesare: differential hybridization to cDNA libraries; subtractivehybridization; differential display; differential 2-D gelelectrophoresis; DNA probe arrays; and isolation of proteins known to beexpressed with some specificity in the target tissue. Such methods arewell known to those of skill in the art. Commercially available productsfor identifying promoters are known in the art such as the Clontech(Palo Alto, Calif.) Universal GenomeWalker Kit.

[0237] For the protein-based methods, it is helpful to obtain the aminoacid sequence for at least a portion of the identified protein, and thento use the protein sequence as the basis for preparing a nucleic acidthat can be used as a probe to identify either genomic DNA directly, orpreferably, to identify a cDNA clone from a library prepared from thetarget tissue. Once such a cDNA clone has been identified, that sequencecan be used to identify the sequence at the 5′ end of the transcript ofthe indicated gene. For differential hybridization, subtractivehybridization and differential display, the nucleic acid sequenceidentified as enriched in the target tissue is used to identify thesequence at the 5′ end of the transcript of the indicated gene. Oncesuch sequences are identified, starting either from protein sequences ornucleic acid sequences, any of these sequences identified as being fromthe gene transcript can be used to screen a genomic library preparedfrom the target organism. Methods for identifying and confirming thetranscriptional start site are well known in the art.

[0238] In the process of isolating promoters expressed under particularenvironmental conditions or stresses, or in specific tissues, or atparticular developmental stages, a number of genes are identified thatare expressed under the desired circumstances, in the desired tissue, orat the desired stage. Further analysis will reveal expression of eachparticular gene in one or more other tissues of the plant. One canidentify a promoter with activity in the desired tissue or condition butthat do not have activity in any other common tissue.

[0239] To identify the promoter sequence, the 5′ portions of the clonesdescribed here are analyzed for sequences characteristic of promotersequences. For instance, promoter sequence elements include the TATA boxconsensus sequence (TATAAT), which is usually an AT-rich stretch of 5-10bp located approximately 20 to 40 base pairs upstream of thetranscription start site. Identification of the TATA box is well knownin the art. For example, one way to predict the location of this elementis to identify the transcription start site using standard RNA-mappingtechniques such as primer extension, S1 analysis, and/or RNaseprotection. To confirm the presence of the AT-rich sequence, astructure-function analysis can be performed involving mutagenesis ofthe putative region and quantification of the mutation's effect onexpression of a linked downstream reporter gene. See, e.g., The MaizeHandbook, Chapter 114, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0240] In plants, further upstream from the TATA box, at positions −80to −100, there is typically a promoter element (i.e., the CAAT box) witha series of adenines surrounding the trinucleotide G (or T) N G. J.Messing et al., in Genetic Engineering in Plants, Kosage, Meredith andHollaender, Eds., pp. 221-227 (1983). In maize, there is no wellconserved CAAT box but there are several short, conservedprotein-binding motifs upstream of the TATA box. These include motifsfor the trans-acting transcription factors involved in light regulation,anaerobic induction, hormonal regulation, or anthocyanin biosynthesis,as appropriate for each gene.

[0241] Once promoter and/or gene sequences are known, a region ofsuitable size is selected from the genomic DNA that is 5′ to thetranscriptional start, or the translational start site, and suchsequences are then linked to a coding sequence. If the transcriptionalstart site is used as the point of fusion, any of a number of possible5′ untranslated regions can be used in between the transcriptional startsite and the partial coding sequence. If the translational start site atthe 3′ end of the specific promoter is used, then it is linked directlyto the methionine start codon of a coding sequence.

[0242] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0243] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold. Buchman and Berg,Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Such intron enhancement of gene expression istypically greatest when placed near the 5′ end of the transcriptionunit. Use of maize introns Adh1-S intron 1, 2, and 6, the Bronze-1intron are known in the art. See generally, The Maize Handbook, Chapter116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0244] The vector comprising the sequences from a polynucleotide of thepresent invention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker genewill encode antibiotic resistance, with suitable genes including genescoding for resistance to the antibiotic spectinomycin (e.g., the aadagene), the streptomycin phosphotransferase (SPT) gene coding forstreptomycin resistance, the neomycin phosphotransferase (NPTII) geneencoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance, genescoding for resistance to herbicides which act to inhibit the action ofacetolactate synthase (ALS), in particular the sulfonylurea-typeherbicides (e.g., the acetolactate synthase (ALS) gene containingmutations leading to such resistance in particular the S4 and/or Hramutations), genes coding for resistance to herbicides which act toinhibit action of glutamine synthase, such as phosphinothricin or basta(e.g., the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta, the nptll gene encodesresistance to the antibiotics kanamycin and geneticin, and the ALS geneencodes resistance to the herbicide chlorsulfuron.

[0245] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol. 153:253-277 (1987). These vectors areplant integrating vectors in that on transformation, the vectorsintegrate a portion of vector DNA into the genome of the host plant.Exemplary A. tumefaciens vectors useful herein are plasmids pKYLX6 andpKYLX7 of Schardl et al., Gene 61:1-11 (1987) and Berger et al., Proc.Natl. Acad. Sci. U.S.A. 86:8402-8406 (1989). Another useful vectorherein is plasmid pBI101.2 that is available from Clontech Laboratories,Inc. (Palo Alto, Calif.).

[0246] A polynucleotide of the present invention can be expressed ineither sense or anti-sense orientation as desired. It will beappreciated that control of gene expression in either sense oranti-sense orientation can have a direct impact on the observable plantcharacteristics. Antisense technology can be conveniently used to geneexpression in plants. To accomplish this, a nucleic acid segment fromthe desired gene is cloned and operably linked to a promoter such thatthe anti-sense strand of RNA will be transcribed. The construct is thentransformed into plants and the antisense strand of RNA is produced. Inplant cells, it has been shown that antisense RNA inhibits geneexpression by preventing the accumulation of mRNA which encodes theenzyme of interest, see, e.g., Sheehy et al., Proc. Nat'l. Acad. Sci.USA 85:8805-8809 (1988); and Hiatt et al., U.S. Pat. No. 4,801,340.

[0247] Another method of suppression is sense suppression. Introductionof nucleic acid configured in the sense orientation has been shown to bean effective means by which to block the transcription of target genes.For an example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323.

[0248] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. It is possible to design ribozymes thatspecifically pair with virtually any target RNA and cleave thephosphodiester backbone at a specific location, thereby functionallyinactivating the target RNA. In carrying out this cleavage, the ribozymeis not itself altered, and is thus capable of recycling and cleavingother molecules, making it a true enzyme. The inclusion of ribozymesequences within antisense RNAs confers RNA-cleaving activity upon them,thereby increasing the activity of the constructs. The design and use oftarget RNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

[0249] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res.(1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R.B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home, et al., J. Am. Chem. Soc. (1990) 112:2435-2437.Use of N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been described by Webb andMatteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res.(1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and, 5,681941.

[0250] Proteins

[0251] The isolated proteins of the present invention comprise apolypeptide having at least 10 amino acids encoded by any one of thepolynucleotides of the present invention as discussed more fully, supra,or polypeptides which are conservatively modified variants thereof.Exemplary polypeptide sequences are provided in SEQ ID NOS: 2, 12, 14,or 22. The proteins of the present invention or variants thereof cancomprise any number of contiguous amino acid residues from a polypeptideof the present invention, wherein that number is selected from the groupof integers consisting of from 10 to the number of residues in afull-length cell cycle polypeptide. Optionally, this subsequence ofcontiguous amino acids is at least 15, 20, 25, 30, 35, or 40 amino acidsin length, often at least 50, 60, 70, 80, or 90 amino acids in length.Further, the number of such subsequences can be any integer selectedfrom the group consisting of from 1 to 20, such as 2, 3, 4, or 5.

[0252] As those of skill will appreciate, the present invention includescatalytically active polypeptides of the present invention (i.e.,enzymes). Catalytically active polypeptides have a specific activity atleast 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, andmost preferably at least 80%, 90%, or 95% that of the native(non-synthetic), endogenous polypeptide. Further, the substratespecificity (k_(cat)/K_(m)) is optionally substantially similar to thenative (non-synthetic), endogenous polypeptide. Typically, the K_(m)will be at least 30%, 40%, or 50%, that of the native (non-synthetic),endogenous polypeptide; and more preferably at least 60%, 70%, 80%, or90%. Methods of assaying and quantifying measures of enzymatic activityand substrate specificity (k_(cat)/K_(m)), are well known to those ofskill in the art.

[0253] Generally, the proteins of the present invention will, whenpresented as an immunogen, elicit production of an antibody specificallyreactive to a polypeptide of the present invention encoded by apolynucleotide of the present invention as described, supra. Exemplarypolypeptides include those which are full-length, such as thosedisclosed in SEQ ID NOS: 2, 12, 14, or 22. Further, the proteins of thepresent invention will not bind to antisera raised against a polypeptideof the present invention which has been fully immunosorbed with the samepolypeptide. Immunoassays for determining binding are well known tothose of skill in the art. A preferred immunoassay is a competitiveimmunoassay as discussed, infra. Thus, the proteins of the presentinvention can be employed as immunogens for constructing antibodiesimmunoreactive to a protein of the present invention for such exemplaryutilities as immunoassays or protein purification techniques.

[0254] Expression of Proteins in Host Cells

[0255] Using the nucleic acids of the present invention, one may expressa protein of the present invention in a recombinantly engineered cellsuch as bacteria, yeast, insect, mammalian, or preferably plant cells.The cells produce the protein in a non-natural condition (e.g., inquantity, composition, location, and/or time), because they have beengenetically altered through human intervention to do so. In eukaryoticcells overexpression of a non-functional fusion protein may bedesirable. After isolation and purification of the fusion protein fromthe expressing cells, enzymatic cleavage could be used to restorefunction to the purified CycD protein. In addition, fusions with CycDcan have application for affinity matrices and affinity columns used forpurifying other cell cycle genes. For example, “His-patch” thioredoxinfusions can be expressed, and the isolate His-CycD fusion protein boundto metal chelate columns. Whole cell protein extracts can then be passedthrough the column to selectively trap proteins that interact with CycD.See Ausubel et al., 1990 for general methods. Similarly, glutathione-Stransferase fusions can be used to attach proteins to solid-phasematrices for this type of affinity binding. This method has been used,for example, to identify cell cycle genes whose proteins bind to GST-Rbin L. Magnaghi-Jaulin et al., Retinoblastoma protein repressestranscription by recruiting a histone deacetylase. Nature 391:601-604(1998). It may also be advantageous to fuse additional functional genesto the CycD gene. For example it would be useful to fuse a greenfluorescent gene or some other reporter gene.

[0256] It is expected that those of skill in the art are knowledgeablein the numerous expression systems available for expression of a nucleicacid encoding a protein of the present invention. No attempt to describein detail the various methods known for the expression of proteins inprokaryotes or eukaryotes will be made.

[0257] In brief summary, the expression of isolated nucleic acidsencoding a protein of the present invention will typically be achievedby operably linking, for example, the DNA or cDNA to a promoter (whichis either constitutive or inducible) followed by incorporation into anexpression vector. The vectors can be suitable for replication andintegration in either prokaryotes or eukaryotes. Typical expressionvectors contain transcription and translation terminators, initiationsequences, and promoters useful for regulation of the expression of theDNA encoding a protein of the present invention. To obtain high levelexpression of a cloned gene, it is desirable to construct expressionvectors which contain, at the minimum, a strong promoter to directtranscription, a ribosome binding site for translational initiation, anda transcription/translation terminator. One of skill would recognizethat modifications can be made to a protein of the present inventionwithout diminishing its biological activity. Some modifications may bemade to facilitate the cloning, expression, or incorporation of thetargeting molecule into a fusion protein. Such modifications are wellknown to those of skill in the art and include, for example, amethionine added at the amino terminus to provide an initiation site, oradditional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located restriction sites or termination codons orpurification sequences.

[0258] A. Expression in Prokaryotes

[0259] Prokaryotic cells may be used as hosts for expression.Prokaryotes most frequently are represented by various strains of E.coli; however, other microbial strains may also be used. Commonly usedprokaryotic control sequences which are defined herein to includepromoters for transcription initiation, optionally with an operator,along with ribosome binding site sequences, include such commonly usedpromoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0260] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Appropriate bacterial cells are infected with phage vector particles ortransfected with naked phage vector DNA. If a plasmid vector is used,the bacterial cells are transfected with the plasmid vector DNA.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

[0261] B. Expression in Eukaryotes

[0262] A variety of eukaryotic expression systems such as yeast, insectcell lines, plant and mammalian cells, are known to those of skill inthe art. As explained briefly below, a of the present invention can beexpressed in these eukaryotic systems. In some embodiments,transformed/transfected plant cells, as discussed infra, are employed asexpression systems for production of the proteins of the instantinvention.

[0263] Synthesis of heterologous proteins in yeast is well known.Sherman, F., et al., Methods in Yeast Genetics, Cold Spring HarborLaboratory (1982) is a well recognized work describing the variousmethods available to produce the protein in yeast. Suitable vectorsusually have expression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired. Forinstance, suitable vectors are described in the literature (Botstein etal., Gene 8:17-24 (1979); Broach et al., Gene 8:121-133 (1979)).

[0264] A protein of the present invention, once expressed, can beisolated from yeast by lysing the cells and applying standard proteinisolation techniques to the lysates. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay of other standard immunoassay techniques.

[0265] The sequences encoding proteins of the present invention can alsobe ligated to various expression vectors for use in transfecting cellcultures of, for instance, mammalian, insect, or plant origin.Illustrative of cell cultures useful for the production of the peptidesare mammalian cell cultures. Mammalian cell systems often will be in theform of monolayers of cells although mammalian cell suspensions may alsobe used. A number of suitable host cell lines capable of expressingintact proteins have been developed in the art, and include the HEK293,BHK21, and CHO cell lines. Expression vectors for these cells caninclude expression control sequences, such as an origin of replication,a promoter (e.g., the CMV promoter, a HSV tk promoter or pgk(phosphoglycerate kinase) promoter), an enhancer (Queen et al., Immunol.Rev. 89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites (e.g.,an SV40 large T Ag poly A addition site), and transcriptional terminatorsequences. Other animal cells useful for production of proteins of thepresent invention are available, for instance, from the American TypeCulture Collection Catalogue of Cell Lines and Hybridomas (7th edition,1992).

[0266] Appropriate vectors for expressing proteins of the presentinvention in insect cells are usually derived from the SF9 baculovirus.Suitable insect cell lines include mosquito larvae, silkworm, armyworm,moth and Drosophila cell lines such as a Schneider cell line (SeeSchneider, J. Embryol. Exp. Morphol. 27:353-365 (1987)).

[0267] As with yeast, when higher animal or plant host cells areemployed, polyadenlyation or transcription terminator sequences aretypically incorporated into the vector. An example of a terminatorsequence is the polyadenlyation sequence from the bovine growth hormonegene. Sequences for accurate splicing of the transcript may also beincluded. An example of a splicing sequence is the VP1 intron from SV40(Sprague et al., J. Virol. 45:773-781 (1983)). Additionally, genesequences to control replication in the host cell may be incorporatedinto the vector such as those found in bovine papilloma virustype-vectors. Saveria-Campo, M., Bovine Papilloma Virus DNA a EukaryoticCloning Vector in DNA Cloning Vol. II a Practical Approach, D. M.Glover, Ed., IRL Press, Arlington, Va., pp. 213-238 (1985).

[0268] Use in Two-Hybrid Systems

[0269] An important utility for the maize CycD genes that have beencloned in the genetic approach of using a two-hybrid system to identifyinteracting proteins (i.e. proteins that specifically interact with theCycD gene-encoded products. This method, typically done using the yeastSaccharomyces cerevisiae, exploits the fact that a functionaltranscription factor can be separated into two components; a DNA-bindingfactor and an activation domain, which when held together non-covalentlywill still bind DNA and activate transcription. The test system isconstructed as follows: a DNA-binding domain is localized 5′ to areporter gene, for example luciferase, and this cassette is transformedinto a yeast strain. The nucleic acid sequence for the DNA-bindingdomain of the transcriptional factor is ligated to the gene (or partialgene sequence) being used as bait. Expression of this DNA-bindingdomain-bait fusion is driven, for example by the yeast adh1 promoter. A“library” of gene-fusions is also produced, using the activation domainof the transcriptional factor fused to genes (or gene fragments) from anexpression library of interest (referred to as the activation domainhybrid). Expression of the activation domain hybrids is alsoaccomplished, for example, using the yeast adh1 promoter. To perform thetwo-hybrid screen, plasmids encoding the DNA-binding domain hybrid and alibrary of activation domain hybrids are introduced (sequentially orsimultaneously) into a yeast strain already containing the inactivereporter. Transformed yeast in which the activation domain hybridspecifically bind to the DNA-binding domain hybrid will expressluciferase. Positives are further characterized by sequence analysis,and further tests of relevance of biological interactions.

[0270] Commonly used DNA-binding domains include those from lexa proteinin E.coli, and the Ga14 protein in yeast. Likewise, commonly usedactivation domains include B42 (bacterial) and Ga14 (yeast). Fordetails, see Hannon G, and Bartel P, Identification of interactingproteins using the two-hybrid system, Methods Mol. Cellular Biol.5:289-297 (1995).

[0271] Transfection/Transformation of Cells

[0272] The method of transformation/transfection is not critical to theinstant invention; various methods of transformation or transfection arecurrently available. As newer methods are available to transform cropsor other host cells they-may be directly applied. Accordingly, a widevariety of methods have been developed to insert a DNA sequence into thegenome of a host cell to obtain the transcription and/or translation ofthe sequence to effect phenotypic changes in the organism. Thus, anymethod which provides for efficient transformation/transfection may beemployed.

[0273] A. Plant Transformation

[0274] A DNA sequence coding for the desired polynucleotide of thepresent invention, for example a cDNA or a genomic sequence encoding afull length protein, will be used to construct a recombinant expressioncassette which can be introduced into the desired plant.

[0275] Gene Transformation Methods

[0276] Numerous methods for introducing foreign genes into plants areknown and can be used to insert the cell cycle gene into a plant host,including biological and; physical plant transformation protocols. See,for example, Miki et.al., 1993, “Procedure for Introducing Foreign DNAinto Plants,” In: Methods in Plant Molecular Biology and Biotechnology,Glick and Thompson, eds., CRC Press, Inc., Boca Raton, pages 67-88. Themethods chosen vary with the host plant, and include chemicaltransfection methods such as calcium phosphate, microorganism-mediatedgene transfer such as Agrobacterium (Horsch et al., Science 227:1229-31,1985), electroporation, micro-injection, and biolistic bombardment.

[0277] Expression cassettes and vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants are knownand available. See, for example, Gruber et.al., 1993, “Vectors for PlantTransformation” In: Methods in Plant Molecular Biology andBiotechnology, Glick and Thompson, eds., CRC Press, Inc., Boca Raton,pages 89-119.

[0278] Agrobacterium-Mediated Transformation

[0279] The most widely utilized method for introducing an expressionvector into plants is based on the natural transformation system ofAgrobacterium. A. tumefaciens and A. rhizogenes are plant pathogenicsoil bacteria which genetically transform plant cells. The Ti and Riplasmids of A. tumefaciens and A. rhizogenes, respectfully, carry genesresponsible for genetic transformation of plants. See, for example,Kado, 1991, Crit. Rev. Plant Sci. 10:1. Descriptions of theAgrobacterium vector systems and methods for Agrobacterium-mediated genetransfer are provide in Gruber et al., supra; Miki et al., supra; andMoloney et al., 1989, Plant Cell Reports 8:238.

[0280] Direct Gene Transfer

[0281] Methods for Agrobacterium-mediated transformation in rice isdisclosed in (Hiei et.al., 1994, The Plant Journal 6:271-282) and maize(Ishida et al., 1996, Nature/Biotechnology 14:745-750). Several methodsof plant transformation, collectively referred to as direct genetransfer, have been developed as an alternative toAgrobacterium-mediated transformation. Methods forAgrobacterium-mediated transformation in sorghum are disclosed in WO98/49332. Methods for Agrobacterium-mediated transformation in maize aredisclosed in WO 98/32326.

[0282] A generally applicable method of plant transformation ismicroprojectile-mediated transformation, where DNA is carried on thesurface of microprojectiles measuring about 1 to 4 μm. The expressionvector is introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate the plant cell walls and membranes. (Sanford etal., 1987, Part. Sci. Technol. 5:27; Sanford, 1988, Trends Biotech6:299; Sanford, 1990, Physiol. Plant 79:206; Klein et al., 1992,Biotechnology 10:268).

[0283] Another method for physical delivery of DNA to plants issonication of target cells as described in Zang et al., 1991,Bio/Technology 9:996. Alternatively, liposome or spheroplast fusionshave been used to introduce expression vectors into plants. See, forexample, Deshayes et al., 1985, EMBO J. 4:2731; and Christou et al.,1987, PNAS USA 84:3962. Direct uptake of DNA into protoplasts usingCaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine have alsobeen reported. See, for example, Hain et al., 1985, Mol. Gen.Genet.199:161; and Draper et al., 1982, Plant Cell Physiol. 23:451.

[0284] Electroporation of protoplasts and whole cells and tissues hasalso been described. See, for example, Donn et al., 1990, In: Abstractsof the Vllth Int'l Congress on Plant Cell and Tissue Culture (IAPTC),A2-38, page 53; D'Halluin et al., 1992, Plant Cell 4:1495-1505; andSpencer et al., 1994, Plant Mol.Biol. 24:51-61. Microinjection of DNAinto whole plant cells has also been described as has microinjectioninto protoplasts. See, for example in whole cells, Neuhaus et al., 1987,Theor. Appl. Genet. 75:30-36; and in protoplasts, Crossway et al., 1986,Mol. Gen. Genet. 202:179-185; and Reich et al., 1986, Biotechnology4:1001-1004.

[0285] Particle Wounding/Agrobacterium Delivery

[0286] Another useful basic transformation protocol involves acombination of wounding by particle bombardment, followed by use ofAgrobacterium for DNA delivery, as described by Bidney et al., PlantMol. Biol. 18:301-313 (1992). Useful plasmids for plant transformationinclude PHP9762. The binary backbone for PHP9762 is bin 19. See Bevan,Nucleic Acids Research 12:8711-8721 (1984).

[0287] In general, the intact meristem transformation method involvesimbibing seed for 24 hours in the dark, removing the cotyledons and rootradical, followed by culturing of the meristem explants. Twenty-fourhours later, the primary leaves are removed to expose the apicalmeristem. The explants are placed apical dome side up and bombarded,e.g., twice with particles, followed by co-cultivation withAgrobacterium. To start the co-cultivation for intact meristems,Agrobacterium is placed on the meristem. After about a 3-dayco-cultivation period the meristems are transferred to culture mediumwith cefotaxime (plus kanamycin for the NPTII selection). Selection canalso be done using kanamycin.

[0288] The split meristem method involves imbibing seed, breaking of thecotyledons to produce a clean fracture at the plane of the embryonicaxis, excising the root tip and then bisecting the explantslongitudinally between the primordial leaves. The two halves are placedcut surface up on the medium then bombarded twice with particles,followed by co-cultivation with Agrobacterium. For split meristems,after bombardment, the meristems are placed in an Agrobacteriumsuspension for 30 minutes. They are then removed from the suspensiononto solid culture medium for three day co-cultivation. After thisperiod, the meristems are transferred to fresh medium with cefotaxime(plus kanamycin for selection).

[0289] Transfer by Plant Breeding

[0290] Once a single transformed plant has been obtained by theforegoing recombinant DNA method, e.g., a plant transformed with adesired gene, conventional plant breeding methods can be used totransfer the structural gene and associated regulatory sequences viacrossing and backcrossing. In general, such plant breeding techniquesare used to transfer a desired gene into a specific crop plant. In theinstant invention, such methods include the further steps of: (1)sexually crossing a transformed plant with a second non-transformedplant; (2) recovering reproductive material from the progeny of thecross; and (3) growing transformed containing plants from thereproductive material.

[0291] Isolated nucleic acid acids of the present invention can beintroduced into plants according techniques known in the art. Generally,recombinant expression cassettes as described above and suitable fortransformation of plant cells are prepared. Techniques for transforminga wide variety of higher plant species are well known and described inthe technical, scientific, and patent literature. See, for example,Weising et al., Ann. Rev. Genet. 22:421-477 (1988). For example, the DNAconstruct may be introduced directly into the genomic DNA of the plantcell using techniques such as electroporation, PEG poration, particlebombardment, silicon fiber delivery, or microinjection of plant cellprotoplasts or embryogenic callus. Alternatively, the DNA constructs maybe combined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria.

[0292] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. 82:5824 (1985). Ballistic transformation techniques aredescribed in Klein et al., Nature 327:70-73 (1987).

[0293]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). Although Agrobacterium is useful primarily indicots, certain monocots can be transformed by Agrobacterium. Forinstance, Agrobacterium transformation of maize is described in U.S.Pat. No. 5,550,318.

[0294] Other methods of transfection or transformation include (1)Agrobacterium rhizogenes-mediated transformation (see, e.g.,Lichtenstein and Fuller In: Genetic Engineering, vol. 6, PWJ Rigby, Ed.,London, Academic Press, 1987; and Lichtenstein, C. P., and Draper, J,.In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985),Application PCT/US87/02512 (WO 88/02405 published Apr. 7, 1988)describes the use of A.rhizogenes strain A4 and its Ri plasmid alongwith A. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNAuptake (see, e.g., Freeman et al., Plant Cell Physiol. 25:1353, 1984),(3) the vortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA87:1228, (1990)).

[0295] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlaneMol. Biol. Reporter 6:165 (1988). Expression of polypeptide coding genescan be obtained by injection of the DNA into reproductive organs of aplant as described by Pena et al., Nature 325:274 (1987). DNA can alsobe injected directly into the cells of immature embryos and therehydration of desiccated embryos as described by Neuhaus et al., Theor.Appl. Genet., 75:30 (1987); and Benbrook et al., in Proceedings Bio Expo1986, Butterworth, Stoneham, Mass., pp. 27-54 (1986). A variety of plantviruses that can be employed as vectors are known in the art and includecauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, andtobacco mosaic virus.

[0296] B. Transfection of Prokaryotes, Lower Eukaryotes, and AnimalCells

[0297] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transfection by various means. Thereare several well-known methods of introducing DNA into animal cells.These include: calcium phosphate precipitation, fusion of the recipientcells with bacterial protoplasts containing the DNA, treatment of therecipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

[0298] Synthesis of Proteins

[0299] The proteins of the present invention can be constructed usingnon-cellular synthetic methods. Solid phase synthesis of proteins ofless than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicycylohexylcarbodiimide) is known to those of skill.

[0300] Purification of Proteins

[0301] The proteins of the present invention may be purified by standardtechniques well known to those of skill in the art. Recombinantlyproduced proteins of the present invention can be directly expressed orexpressed as a fusion protein. The recombinant protein is purified by acombination of cell lysis (e.g., sonication, French press) and affinitychromatography. For fusion products, subsequent digestion of the fusionprotein with an appropriate proteolytic enzyme releases the desiredrecombinant protein.

[0302] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including selective precipitation with such substances as ammoniumsulfate, column chromatography, immunopurification methods, and others.See, for instance, R. Scopes, Protein Purification: Principles andPractice, Springer-Verlag: New York (1982); Deutscher, Guide to ProteinPurification, Academic Press (1990). For example, antibodies may beraised to the proteins as described herein. Purification from E. colican be achieved following procedures described in U.S. Pat. No.4,511,503. The protein may then be isolated from cells expressing theprotein and further purified by standard protein chemistry techniques asdescribed herein. Detection of the expressed protein is achieved bymethods known in the art and include, for example, radioimmunoassays,Western blotting techniques or immunoprecipitation.

[0303] Transgenic Plant Regeneration

[0304] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerwhich has been introduced together with a polynucleotide of the presentinvention.

[0305] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillilan Publishing Company, New York, pp. 124-176 (1983); andBinding, Regeneration of Plants, Plant Protoplasts, CRC Press, BocaRaton, pp. 21-73 (1985).

[0306] Transformed plant cells, calli or explant can be cultured onregeneration medium in the dark for several weeks, generally about 1 to3 weeks to allow the somatic embryos to mature. Preferred regenerationmedia include media containing MS salts, such as PHI-E and PHI-F media.The plant cells, calli or explant are then typically cultured on rootingmedium in a light/dark cycle until shoots and roots develop. Methods forplant regeneration are known in the art and preferred methods areprovided by Kamo et al., (Bot. Gaz. 146(3):324-334, 1985), West et al.,(The Plant Cell 5:1361-1369, 1993), and Duncan et al. (Planta165:322-332, 1985).

[0307] Small plantlets can then be transferred to tubes containingrooting medium and allowed to grow and develop more roots forapproximately another week. The plants can then be transplanted to soilmixture in pots in the greenhouse.

[0308] The regeneration of plants containing the foreign gene introducedby Agrobacterium from leaf explants can be achieved as described byHorsch et al., Science 227:1229-1231 (1985). In this procedure,transformants are grown in the presence of a selection agent and in amedium that induces the regeneration of shoots in the plant speciesbeing transformed as described by Fraley et al., Proc. Natl. Acad. Sci.U.S.A. 80:4803 (1983). This procedure typically produces shoots withintwo to four weeks and these transformant shoots are then transferred toan appropriate root-inducing medium containing the selective agent andan antibiotic to prevent bacterial growth. Transgenic plants of thepresent invention may be fertile or sterile.

[0309] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). This regeneration and growthprocess includes the steps of selection of transformant cells andshoots, rooting the transformant shoots and growth of the plantlets insoil. For maize cell culture and regeneration see generally, The MaizeHandbook, Freeling and Walbot, Eds., Springer, New York (1994); Corn andCorn Improvement, 3^(rd) edition, Sprague and Dudley Eds., AmericanSociety of Agronomy, Madison, Wis. (1988).

[0310] One of skill will recognize that after the recombinant expressioncassette is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

[0311] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype, (e.g., altered cell cycle content or composition).

[0312] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

[0313] Transgenic plants expressing the selectable marker can bescreened for transmission of the nucleic acid of the present inventionby, for example, standard immunoblot and DNA detection techniques.Transgenic lines are also typically evaluated on levels of expression ofthe heterologous nucleic acid. Expression at the RNA level can bedetermined initially to identify and quantitate expression-positiveplants. Standard techniques for RNA analysis can be employed and includePCR amplification assays using oligonucleotide primers designed toamplify only the heterologous RNA templates and solution hybridizationassays using heterologous nucleic acid-specific probes. The RNA-positiveplants can then analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

[0314] A preferred embodiment is a transgenic plant that is homozygousfor the added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered cell division relative to a control plant (i.e., native,non-transgenic). Back-crossing to a parental plant and out-crossing witha non-transgenic plant are also contemplated.

[0315] Modulating Cell Cycle Protein Content and/or Composition

[0316] The present invention further provides a method for modulating(i.e., increasing or decreasing) cell cycle protein content orcomposition in a plant or part thereof. Modulation can be effected byincreasing or decreasing the cell cycle protein content (i.e., the totalamount of cell cycle protein) and/or the cell cycle protein composition(the ratio of various cell cycle monomers in the plant) in a plant. Themethod comprises transforming a plant cell, transiently or stably, witha recombinant expression cassette comprising a polynucleotide of thepresent invention as described above to obtain a transformed plant cell.For stably transformed plant cells, growing the transformed plant cellunder plant forming conditions, and inducing expression of apolynucleotide of the present invention in the plant for a timesufficient to modulate cell cycle protein content and/or composition inthe plant or plant part.

[0317] In some embodiments, plant cell division may be modulated byaltering, in vivo or in vitro, the promoter of a non-isolated cell cyclegene to up- or down-regulate gene expression. In some embodiments, thecoding regions of native cell cycle genes can be altered viasubstitution, addition, insertion, or deletion to decrease activity ofthe encoded enzyme. See, e.g., Kmiec, U.S. Pat. No. 5,565,350; Zarlinget al., PCT/US93/03868. And in some embodiments, an isolated nucleicacid (e.g., a vector) comprising a promoter sequence is transfected intoa plant cell. Subsequently, a plant cell comprising the promoteroperably linked to a polynucleotide of the present invention is selectedfor by means known to those of skill in the art such as, but not limitedto, Southern blot, DNA sequencing, or PCR analysis using primersspecific to the promoter and to the gene and detecting ampliconsproduced therefrom. A plant or plant part altered or modified by theforegoing embodiments is grown under plant forming conditions for a timesufficient to modulate cell cycle protein content and/or composition inthe plant. Plant forming conditions are well known in the art anddiscussed briefly, supra.

[0318] In general, content or composition is increased or decreased byat least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relative toa native control plant, plant part, or cell lacking the aforementionedrecombinant expression cassette. Modulation in the present invention mayoccur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds that activate expression from these promoters arewell known in the art. In preferred embodiments, cell division ismodulated in monocots, particularly maize.

[0319] Molecular Markers

[0320] The present invention provides a method of genotyping a plantcomprising a polynucleotide of the present invention. Genotypingprovides a means of distinguishing homologs of a chromosome pair and canbe used to differentiate segregants in a plant population. Molecularmarker methods can be used for phylogenetic studies, characterizinggenetic relationships among crop varieties, identifying crosses orsomatic hybrids, localizing chromosomal segments affecting monogenictraits, map based cloning, and the study of quantitative inheritance.See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7,Clark, Ed., Springer-Verlag, Berlin (1997). For molecular markermethods, see generally, The DNA Revolution by Andrew H. Paterson 1996(Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) byAcademic Press/R. G. Landis Company, Austin, Tex., pp.7-21.

[0321] The particular method of genotyping in the present invention mayemploy any number of molecular marker analytic techniques such as, butnot limited to, restriction fragment length polymorphisms (RFLPs). RFLPsare the product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. As is well known to those ofskill in the art, RFLPs are typically detected by extraction of genomicDNA and digestion with a restriction enzyme. Generally, the resultingfragments are separated according to size and hybridized with a probe;single copy probes are preferred. Restriction fragments from homologouschromosomes are revealed. Differences in fragment size among allelesrepresent an RFLP. Thus, the present invention further provides a meansto follow segregation of a cell cycle gene or nucleic acid of thepresent invention as well as chromosomal sequences genetically linked tothese genes or nucleic acids using such techniques as RFLP analysis.Linked chromosomal sequences are within 50 centiMorgans (cM), oftenwithin 40 or 30 cM, preferably within 20 or 10 cM, more preferablywithin 5, 3, 2, or 1 cM of a cell cycle gene.

[0322] In the present invention, the nucleic acid probes employed formolecular marker mapping of plant nuclear genomes selectively hybridize,under selective hybridization conditions, to a gene encoding apolynucleotide of the present invention. In preferred embodiments, theprobes are selected from polynucleotides of the present invention.Typically, these probes are cDNA probes or Pst I genomic clones. Thelength of the probes is discussed in greater detail, supra, but aretypically at least 15 bases in length, more preferably at least 20, 25,30, 35, 40, or 50 bases in length. Generally, however, the probes areless than about 1 kilobase in length. Preferably, the probes are singlecopy probes that hybridize to a unique locus in a haploid chromosomecomplement. Some exemplary restriction enzymes employed in RFLP mappingare EcoRI, EcoRv, and Sstl. As used herein the term “restriction enzyme”includes reference to a composition that recognizes and, alone or inconjunction with another composition, cleaves at a specific nucleotidesequence.

[0323] The method of detecting an RFLP comprises the steps of (a)digesting genomic DNA of a plant with a restriction enzyme; (b)hybridizing a nucleic acid probe, under selective hybridizationconditions, to a sequence of a polynucleotide of the present of thegenomic DNA; (c) detecting therefrom a RFLP. Other methods ofdifferentiating polymorphic (allelic) variants of polynucleotides of thepresent invention can be had by utilizing molecular marker techniqueswell known to those of skill in the art including such techniques as: 1)single stranded conformation analysis (SSCP); 2) denaturing gradient gelelectrophoresis (DGGE); 3) RNase protection assays; 4) allele-specificoligonucleotides (ASOs); 5) the use of proteins which recognizenucleotide mismatches, such as the E. coli mutS protein; and 6)allele-specific PCR. Other approaches based on the detection ofmismatches between the two complementary DNA strands include clampeddenaturing gel electrophoresis (CDGE); heteroduplex analysis (HA); andchemical mismatch cleavage (CMC). Exemplary polymorphic variants areprovided in Table I, supra. Thus, the present invention further providesa method of genotyping comprising the steps of contacting, understringent hybridization conditions, a sample suspected of comprising apolynucleotide of the present invention with a nucleic acid probe.Generally, the sample is a plant sample; preferably, a sample suspectedof comprising a maize polynucleotide of the present invention (e.g.,gene, mRNA). The nucleic acid probe selectively hybridizes, understringent conditions, to a subsequence of a polynucleotide of thepresent invention comprising a polymorphic marker. Selectivehybridization of the nucleic acid probe to the polymorphic markernucleic acid sequence yields a hybridization complex. Detection of thehybridization complex indicates the presence of that polymorphic markerin the sample. In preferred embodiments, the nucleic acid probecomprises a polynucleotide of the present invention.

[0324] UTR's and Codon Preference

[0325] In general, translational efficiency has been found to beregulated by specific sequence elements in the 5′ non-coding oruntranslated region (5′ UTR) of the RNA. Positive sequence motifsinclude translational initiation consensus sequences (Kozak, NucleicAcids Res.15:8125 (1987)) and the 5<G>7 methyl GpppG cap structure(Drummond et al., Nucleic Acids Res. 13:7375 (1985)). Negative elementsinclude stable intramolecular 5′ UTR stem-loop structures (Muesing etal., Cell 48:691 (1987)) and AUG sequences or short open reading framespreceded by an appropriate AUG in the 5′ UTR (Kozak, supra, Rao et al.,Mol. and Cell. Biol. 8:284 (1988)). Accordingly, the present inventionprovides 5′ and/or 3′UTR regions for modulation of translation ofheterologous coding sequences.

[0326] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency and/or tooptimize the coding sequence for expression in a desired host or tooptimize the codon usage in a heterologous sequence for expression inmaize. Codon usage in the coding regions of the polynucleotides of thepresent invention can be analyzed statistically using commerciallyavailable software packages such as “Codon Preference” available fromthe University of Wisconsin Genetics Computer Group (see Devereaux etal., Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (EastmanKodak Co., New Haven, Conn.). Thus, the present invention provides acodon usage frequency characteristic of the coding region of at leastone of the polynucleotides of the present invention. The number ofpolynucleotides that can be used to determine a codon usage frequencycan be any integer from 1 to the number of polynucleotides of thepresent invention as provided herein. Optionally, the polynucleotideswill be full-length sequences. An exemplary number of sequences forstatistical analysis can be at least 1, 5, 10, 20, 50, or 100.

[0327] Sequence Shuffling

[0328] The present invention provides methods for sequence shufflingusing polynucleotides of the present invention, and compositionsresulting therefrom. Sequence shuffling is described in PCT publicationNo. 96/19256. See also, Zhang, J. H., et al. Proc. Natl. Acad. Sci. USA94:4504-4509 (1997). Generally, sequence shuffling provides a means forgenerating libraries of polynucleotides having a desired characteristicwhich can be selected or screened for. Libraries of recombinantpolynucleotides are generated from a population of related sequencepolynucleotides which comprise sequence regions which have substantialsequence identity and can be homologously recombined in vitro or invivo. The population of sequence-recombined polynucleotides comprises asubpopulation of polynucleotides which possess desired or advantageouscharacteristics and which can be selected by a suitable selection orscreening method. The characteristics can be any property or attributecapable of being selected for or detected in a screening system, and mayinclude properties of: an encoded protein, a transcriptional element, asequence controlling transcription, RNA processing, RNA stability,chromatin conformation, translation, or other expression property of agene or transgene, a replicative element, a protein-binding element, orthe like, such as any feature which confers a selectable or detectableproperty. In some embodiments, the selected characteristic will be anincreased K_(m) and/or K_(cat) over the wild-type protein as providedherein. In other embodiments, a protein or polynculeotide generated fromsequence shuffling will have a ligand binding affinity greater than thenon-shuffled wild-type polynucleotide. The increase in such propertiescan be at least 110%, 120%, 130%, 140% or at least 150% of the wild-typevalue.

[0329] Detection of Nucleic Acids

[0330] The present invention further provides methods for detecting apolynucleotide of the present invention in a nucleic acid samplesuspected of comprising a polynucleotide of the present invention, suchas a plant cell lysate, particularly a lysate of corn. In someembodiments, a cell cycle gene or portion thereof can be amplified priorto the step of contacting the nucleic acid sample with a polynucleotideof the present invention. The nucleic acid sample is contacted with thepolynucleotide to form a hybridization complex. The polynucleotidehybridizes under stringent conditions to a gene encoding a polypeptideof the present invention. Formation of the hybridization complex is usedto detect a gene encoding a polypeptide of the present invention in thenucleic acid sample. Those of skill will appreciate that an isolatednucleic acid comprising a polynucleotide of the present invention shouldlack cross-hybridizing sequences in common with non-cell cycle genesthat would yield a false positive result.

[0331] Detection of the hybridization complex can be achieved using anynumber of well-known methods. For example, the nucleic acid sample, or aportion thereof, may be assayed by hybridization formats including butnot limited to, solution phase, solid phase, mixed phase, or in situhybridization assays. Briefly, in solution (or liquid) phasehybridizations, both the target nucleic acid and the probe or primer arefree to interact in the reaction mixture. In solid phase hybridizationassays, probes or primers are typically linked to a solid support wherethey are available for hybridization with target nucleic in solution. Inmixed phase, nucleic acid intermediates in solution hybridize to targetnucleic acids in solution as well as to a nucleic acid linked to a solidsupport. In in situ hybridization, the target nucleic acid is liberatedfrom its cellular surroundings in such as to be available forhybridization within the cell while preserving the cellular morphologyfor subsequent interpretation and analysis. The following articlesprovide an overview of the various hybridization assay formats: Singeret al., Biotechniques 4(3):230-250 (1986); Haase et al., Methods inVirology, Vol. VII, pp.189-226 (1984); Wilkinson, The theory andpractice of in situ hybridization in: In situ Hybridization, D. G.Wilkinson, Ed., IRL Press, Oxford University Press, Oxford; and NucleicAcid Hybridization: A Practical Approach, Hames, B. D. and Higgins, S.J., Eds., IRL Press (1987).

[0332] Nucleic Acid Labels and Detection Methods

[0333] The means by which nucleic acids of the present invention arelabeled is not a critical aspect of the present invention and can beaccomplished by any number of methods currently known or laterdeveloped. Detectable labels suitable for use in the present inventioninclude any composition detectable by spectroscopic, radioisotopic,photochemical, biochemical, immunochemical, electrical, optical orchemical means. Useful labels in the present invention include biotinfor staining with labeled streptavidin conjugate, magnetic beads,fluorescent dyes (e.g., fluorescein, Texas red, rhodamine, greenfluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵I, ³⁵S,¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkalinephosphatase and others commonly used in an ELISA), and colorimetriclabels such as colloidal gold or colored glass or plastic (e.g.,polystyrene, polypropylene, latex, etc.) beads.

[0334] Nucleic acids of the present invention can be labeled by any oneof several methods typically used to detect the presence of hybridizednucleic acids. One common method of detection is the use ofautoradiography using probes labeled with ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P, orthe like. The choice of radio-active isotope depends on researchpreferences due to ease of synthesis, stability, and half lives of theselected isotopes. Other labels include ligands which bind to antibodieslabeled with fluorophores, chemiluminescent agents, and enzymes.Alternatively, probes can be conjugated directly with labels such asfluorophores, chemiluminescent agents or enzymes. The choice of labeldepends on sensitivity required, ease of conjugation with the probe,stability requirements, and available instrumentation. Labeling thenucleic acids of the present invention is readily achieved such as bythe use of labeled PCR primers.

[0335] In some embodiments, the label is simultaneously incorporatedduring the amplification step in the preparation of the nucleic acids.Thus, for example, polymerase chain reaction (PCR) with labeled primersor labeled nucleotides will provide a labeled amplification product. Inanother embodiment, transcription amplification using a labelednucleotide (e.g., fluorescein-labeled UTP and/or CTP) incorporates alabel into the transcribed nucleic acids.

[0336] Non-radioactive probes are often labeled by indirect means. Forexample, a ligand molecule is covalently bound to the probe. The ligandthen binds to an anti-ligand molecule that is either inherentlydetectable or covalently bound to a detectable signal system, such as anenzyme, a fluorophore, or a chemiluminescent compound. Enzymes ofinterest as labels will primarily be hydrolases, such as phosphatases,esterases and glycosidases, or oxidoreductases, particularlyperoxidases. Fluorescent compounds include fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc.Chemiluminescers include luciferin, and 2,3-dihydrophthalazinediones,e.g., luminol. Ligands and anti-ligands may be varied widely. Where aligand has a natural anti-ligand, namely ligands such as biotin,thyroxine, and cortisol, it can be used in conjunction with its labeled,naturally-occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0337] Probes can also be labeled by direct conjugation with a label.For example, cloned DNA probes have been coupled directly to horseradishperoxidase or alkaline phosphatase, (Renz. M., and Kurz, K., AColorimetric Method for DNA Hybridization, Nucl. Acids Res. 12:3435-3444(1984)) and synthetic oligonucleotides have been coupled directly withalkaline phosphatase (Jablonski, E., et al., Preparation ofOligodeoxynucleotide-Alkaline Phosphatase Conjugates and Their Use asHybridization Probes, Nuc. Acids. Res. 14:6115-6128 (1986); and Li P.,et al., Enzyme-linked Synthetic Oligonucleotide probes: Non-RadioactiveDetection of Enterotoxigenic Escherichia Coli in Faeca Specimens, Nucl.Acids Res. 15:5275-5287 (1987)).

[0338] Means of detecting such labels are well known to those of skillin the art. Thus, for example, radiolabels may be detected usingphotographic film or scintillation counters, fluorescent markers may bedetected using a photodetector to detect emitted light. Enzymatic labelsare typically detected by providing the enzyme with a substrate anddetecting the reaction product produced by the action of the enzyme onthe substrate, and colorimetric labels are detected by simplyvisualizing the colored label.

[0339] Antibodies to Proteins

[0340] Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-nativeconfigurations. Anti-idiotypic antibodies can also be generated. Personsof skill know many methods of making antibodies. The followingdiscussion is presented as a general overview of the techniquesavailable; however, one of skill will recognize that many variationsupon the following methods are known.

[0341] A number of immunogens are used to produce antibodiesspecifically reactive with a protein of the present invention. Anisolated recombinant, synthetic, or native cell cycle protein of 5 aminoacids in length or greater and selected from a protein encoded by apolynucleotide of the present invention are the preferred immunogens(antigen) for the production of monoclonal or polyclonal antibodies.Those of skill will readily understand that the proteins of the presentinvention are typically denatured, and optionally reduced, prior toformation of antibodies for screening expression libraries or otherassays in which a putative protein of the present invention is expressedor denatured in a non-native secondary, tertiary, or quaternarystructure. Naturally occurring cell cycle polypeptides can be usedeither in pure or impure form.

[0342] The protein of the present invention is then injected into ananimal capable of producing antibodies. Either monoclonal or polyclonalantibodies can be generated for subsequent use in immunoassays tomeasure the presence and quantity of the protein of the presentinvention. Methods of producing polyclonal antibodies are known to thoseof skill in the art. In brief, an immunogen (antigen), preferably apurified protein, a protein coupled to an appropriate carrier (e.g.,GST, keyhole limpet hemanocyanin, etc.), or a protein incorporated intoan immunization vector such as a recombinant vaccinia virus (see, U.S.Pat. No. 4,722,848) is mixed with an adjuvant and animals are immunizedwith the mixture. The animal's immune response to the immunogenpreparation is monitored by taking test bleeds and determining the titerof reactivity to the protein of interest. When appropriately high titersof antibody to the immunogen are obtained, blood is collected from theanimal and antisera are prepared. Further fractionation of the antiserato enrich for antibodies reactive to the protein is performed wheredesired (See, e.g., Coligan, Current Protocols in Immunology,Wiley/Greene, N.Y. (1991); and Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Press, NY (1989)).

[0343] Antibodies, including binding fragments and single chainrecombinant versions thereof, against predetermined fragments of aprotein of the present invention are raised by immunizing animals, e.g.,with conjugates of the fragments with carrier proteins as describedabove. Typically, the immunogen of interest is a protein of at leastabout 5 amino acids, more typically the protein is 10 amino acids inlength, preferably, 15 amino acids in length and more preferably theprotein is 20 amino acids in length or greater. The peptides aretypically coupled to a carrier protein (e.g., as a fusion protein), orare recombinantly expressed in an immunization vector. Antigenicdeterminants on peptides to which antibodies bind are typically 3to 10amino acids in length.

[0344] Monoclonal antibodies are prepared from cells secreting thedesired antibody. Monoclonals antibodies are screened for binding to aprotein from which the immunogen was derived. Specific monoclonal andpolyclonal antibodies will usually have an antibody binding site with anaffinity constant for its cognate monovalent antigen at least between10⁶-10⁷, usually at least 10⁸, preferably at least 10⁹, more preferablyat least 10¹⁰, and most preferably at least 10¹¹ liters/mole.

[0345] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Basic and Clinical Immunology,4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding,Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press,New York, N.Y. (1986); and Kohler and Milstein, Nature 256:495-497(1975). Summarized briefly, this method proceeds by injecting an animalwith an immunogen comprising a protein of the present invention. Theanimal is then sacrificed and cells taken from its spleen, which arefused with myeloma cells. The result is a hybrid cell or “hybridoma”that is capable of reproducing in vitro. The population of hybridomas isthen screened to isolate individual clones, each of which secrete asingle antibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

[0346] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward et al., Nature341:544-546(1989); and Vaughan et al., Nature Biotechnology, 14:309-314(1996)). Alternatively, high avidity human monoclonal antibodies can beobtained from transgenic mice comprising fragments of the unrearrangedhuman heavy and light chain Ig loci (i.e., minilocus transgenic mice).Fishwild et al., Nature Biotech., 14:845-851 (1996). Also, recombinantimmunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567;and Queen et al., Proc. Nat'l Acad. Sci. 86:10029-10033 (1989).

[0347] The antibodies of this invention are also used for affinitychromatography in isolating proteins of the present invention. Columnsare prepared, e.g., with the antibodies linked to a solid support, e.g.,particles, such as agarose, Sephadex, or the like, where a cell lysateis passed through the column, washed, and treated with increasingconcentrations of a mild denaturant, whereby purified protein arereleased.

[0348] The antibodies can be used to screen expression libraries forparticular expression products such as normal or abnormal protein.Usually the antibodies in such a procedure are labeled with a moietyallowing easy detection of presence of antigen by antibody binding.

[0349] Antibodies raised against a protein of the present invention canalso be used to raise anti-idiotypic antibodies. These are useful fordetecting or diagnosing various pathological conditions related to thepresence of the respective antigens.

[0350] Frequently, the proteins and antibodies of the present inventionwill be labeled by joining, either covalently or non-covalently, asubstance that provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

[0351] Protein Immunoassays

[0352] Means of detecting the proteins of the present invention are notcritical aspects of the present invention. In a preferred embodiment,the proteins are detected and/or quantified using any of a number ofwell-recognized immunological binding assays (see, e.g., U.S. Pat. Nos.4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of thegeneral immunoassays, see also Methods in Cell Biology, Vol. 37:Antibodies in Cell Biology, Asai, Ed., Academic Press, Inc. New York(1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, Eds.(1991). Moreover, the immunoassays of the present invention can beperformed in any of several configurations, e.g., those reviewed inEnzyme Immunoassay, Maggio, Ed., CRC Press, Boca Raton, Fla. (1980);Tijan, Practice and Theory of Enzyme Immunoassays, Laboratory Techniquesin Biochemistry and Molecular Biology, Elsevier Science Publishers B.V., Amsterdam (1985); Harlow and Lane, supra; Immunoassay: A PracticalGuide, Chan, Ed., Academic Press, Orlando, Fla. (1987); Principles andPractice of Immunoassays, Price and Newman Eds., Stockton Press, NY(1991); and Non-isotopic Immunoassays, Ngo, Ed., Plenum Press, NY(1988). Immunological binding assays (or immunoassays) typically utilizea “capture agent” to specifically bind to and often immobilize theanalyte (in this case, a protein of the present invention). The captureagent is a moiety that specifically binds to the analyte. In a preferredembodiment, the capture agent is an antibody that specifically binds aprotein(s) of the present invention. The antibody may be produced by anyof a number of means known to those of skill in the art as describedherein.

[0353] Immunoassays also often utilize a labeling agent to specificallybind to and label the binding complex formed by the capture agent andthe analyte. The labeling agent may itself be one of the moietiescomprising the antibody/analyte complex. Thus, the labeling agent may bea labeled protein of the present invention or a labeled antibodyspecifically reactive to a protein of the present invention.Alternatively, the labeling agent may be a third moiety, such as anotherantibody, that specifically binds to the antibody/protein complex.

[0354] In a preferred embodiment, the labeling agent is a secondantibody bearing a label. Alternatively, the second antibody may lack alabel, but it may, in turn, be bound by a labeled third antibodyspecific to antibodies of the species from which the second antibody isderived. The second can be modified with a detectable moiety, such asbiotin, to which a third labeled molecule can specifically bind, such asenzyme-labeled streptavidin.

[0355] Other proteins capable of specifically binding immunoglobulinconstant regions, such as protein A or protein G may also be used as thelabel agent. These proteins are normal constituents of the cell walls ofstreptococcal bacteria. They exhibit a strong non-immunogenic reactivitywith immunoglobulin constant regions from a variety of species (See,generally Kronval et al., J. Immunol. 111: 401-1406 (1973), andAkerstrom et al., J. Immunol. 135:2589-2542 (1985)).

[0356] Throughout the assays, incubation and/or washing steps may berequired after each combination of reagents. Incubation steps can varyfrom about 5 seconds to several hours, preferably from about 5 minutesto about 24 hours. However, the incubation time will depend upon theassay format, analyte, volume of solution, concentrations, and the like.Usually, the assays will be carried out at ambient temperature, althoughthey can be conducted over a range of temperatures, such as 10° C. to40° C.

[0357] While the details of the immunoassays of the present inventionmay vary with the particular format employed, the method of detecting aprotein of the present invention in a biological sample generallycomprises the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, toa protein of the present invention. The antibody is allowed to bind tothe protein under immunologically reactive conditions, and the presenceof the bound antibody is detected directly or indirectly.

[0358] A. Non-Competitive Assay Formats

[0359] Immunoassays for detecting proteins of the present inventioninclude competitive and noncompetitive formats. Noncompetitiveimmunoassays are assays in which the amount of captured analyte (i.e., aprotein of the present invention) is directly measured. In one preferred“sandwich” assay, for example, the capture agent (e.g., an antibodyspecifically reactive, under immunoreactive conditions, to a protein ofthe present invention) can be bound directly to a solid substrate wherethey are immobilized. These immobilized antibodies then capture theprotein present in the test sample. The protein thus immobilized is thenbound by a labeling agent, such as a second antibody bearing a label.Alternatively, the second antibody may lack a label, but it may, inturn, be bound by a labeled third antibody specific to antibodies of thespecies from which the second antibody is derived. The second can bemodified with a detectable moiety, such as biotin, to which a thirdlabeled molecule can specifically bind, such as enzyme-labeledstreptavidin.

[0360] B. Competitive Assay Formats

[0361] In competitive assays, the amount of analyte present in thesample is measured indirectly by measuring the amount of an added(exogenous) analyte (e.g., a protein of the present invention) displaced(or competed away) from a capture agent (e.g., an antibody specificallyreactive, under immunoreactive conditions, to the protein) by theanalyte present in the sample. In one competitive assay, a known amountof analyte is added to the sample and the sample is then contacted witha capture agent that specifically binds a protein of the presentinvention. The amount of protein bound to the capture agent is inverselyproportional to the concentration of analyte present in the sample.

[0362] In a particularly preferred embodiment, the antibody isimmobilized on a solid substrate. The amount of protein bound to theantibody may be determined either by measuring the amount of proteinpresent in a protein/antibody complex, or alternatively by measuring theamount of remaining uncomplexed protein. The amount of protein may bedetected by providing a labeled protein.

[0363] A hapten inhibition assay is another preferred competitive assay.In this assay a known analyte, (such as a protein of the presentinvention) is immobilized on a solid substrate. A known amount ofantibody specifically reactive, under immunoreactive conditions, to theprotein is added to the sample, and the sample is then contacted withthe immobilized protein. In this case, the amount of antibody bound tothe immobilized protein is inversely proportional to the amount ofprotein present in the sample. Again, the amount of immobilized antibodymay be detected by detecting either the immobilized fraction of antibodyor the fraction of the antibody that remains in solution. Detection maybe direct where the antibody is labeled or indirect by the subsequentaddition of a labeled moiety that specifically binds to the antibody asdescribed above.

[0364] C. Generation of Pooled Antisera for Use in Immunoassays

[0365] A protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ IDNOS: 2, 12, 14, or 22, is determined in an immunoassay. The immunoassayuses a polyclonal antiserum which is raised to a polypeptide of thepresent invention (i.e., the immunogenic polypeptide). This antiserum isselected to have low crossreactivity against other proteins and any suchcrossreactivity is removed by immunoabsorbtion prior to use in theimmunoassay (e.g., by immunosorbtion of the antisera with a protein ofdifferent substrate specificity (e.g., a different enzyme) and/or aprotein with the same substrate specificity but of a different form).

[0366] In order to produce antisera for use in an immunoassay, apolypeptide is isolated as described herein. For example, recombinantprotein can be produced in a mammalian or other eukaryotic cell line. Aninbred strain of mice is immunized with the protein of using a standardadjuvant, such as Freund's adjuvant, and a standard mouse immunizationprotocol (see Harlow and Lane, supra). Alternatively, a syntheticpolypeptide derived from the sequences disclosed herein and conjugatedto a carrier protein is used as an immunogen. Polyclonal sera arecollected and titered against the immunogenic polypeptide in animmunoassay, for example, a solid phase immunoassay with the immunogenimmobilized on a solid support. Polyclonal antisera with a titer of 10⁴or greater are selected and tested for their cross reactivity againstpolypeptides of different forms or substrate specificity, using acompetitive binding immunoassay such as the one described in Harlow andLane, supra, at pages 570-573. Preferably, two or more distinct forms ofpolypeptides are used in this determination. These distinct types ofpolypeptides are used as competitors to identify antibodies that arespecifically bound by the polypeptide being assayed for. The competitivepolypeptides can be produced as recombinant proteins and isolated usingstandard molecular biology and protein chemistry techniques as describedherein.

[0367] Immunoassays in the competitive binding format are used forcrossreactivity determinations. For example, the immunogenic polypeptideis immobilized to a solid support. Proteins added to the assay competewith the binding of the antisera to the immobilized antigen. The abilityof the above proteins to compete with the binding of the antisera to theimmobilized protein is compared to the immunogenic polypeptide. Thepercent crossreactivity for the above proteins is calculated, usingstandard calculations. Those antisera with less than 10% crossreactivitywith a distinct form of a polypeptide are selected and pooled. Thecross-reacting antibodies are then removed from the pooled antisera byimmunoabsorbtion with a distinct form of a polypeptide.

[0368] The immunoabsorbed and pooled antisera are then used in acompetitive binding immunoassay as described herein to compare a second“target” polypeptide to the immunogenic polypeptide. In order to makethis comparison, the two polypeptides are each assayed at a wide rangeof concentrations and the amount of each polypeptide required to inhibit50% of the binding of the antisera to the immobilized protein isdetermined using standard techniques. If the amount of the targetpolypeptide required is less than twice the amount of the immunogenicpolypeptide that is required, then the target polypeptide is said tospecifically bind to an antibody generated to the immunogenic protein.As a final determination of specificity, the pooled antisera is fullyimmunosorbed with the immunogenic polypeptide until no binding to thepolypeptide used in the immunosorbtion is detectable. The fullyimmunosorbed antisera is then tested for reactivity with the testpolypeptide. If no reactivity is observed, then the test polypeptide isspecifically bound by the antisera elicited by the immunogenic protein.

[0369] D. Other Assay Formats

[0370] In a particularly preferred embodiment, Western blot (immunoblot)analysis is used to detect and quantify the presence of protein of thepresent invention in the sample. The technique generally comprisesseparating sample proteins by gel electrophoresis on the basis ofmolecular weight, transferring the separated proteins to a suitablesolid support, (such as a nitrocellulose filter, a nylon filter, orderivatized nylon filter), and incubating the sample with the antibodiesthat specifically bind a protein of the present invention. Theantibodies specifically bind to the protein on the solid support. Theseantibodies may be directly labeled or alternatively may be subsequentlydetected using labeled antibodies (e.g., labeled sheep anti-mouseantibodies) that specifically bind to the antibodies.

[0371] E. Quantification of Proteins

[0372] The proteins of the present invention may be detected andquantified by any of a number of means well known to those of skill inthe art. These include analytic biochemical methods such aselectrophoresis, capillary electrophoresis, high performance liquidchromatography (HPLC), thin layer chromatography (TLC), hyperdiffusionchromatography, and the like, and various immunological methods such asfluid or gel precipitin reactions, immunodiffusion (single or double),immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linkedimmunosorbent assays (ELISAs), immunofluorescent assays, and the like.

[0373] F. Reduction of Non-Specific Binding

[0374] One of skill will appreciate that it is often desirable to reducenon-specific binding in immunoassays and during analyte purification.Where the assay involves an antigen, antibody, or other capture agentimmobilized on a solid substrate, it is desirable to minimize the amountof non-specific binding to the substrate. Means of reducing suchnon-specific binding are well known to those of skill in the art.Typically, this involves coating the substrate with a proteinaceouscomposition. In particular, protein compositions such as bovine serumalbumin (BSA), nonfat powdered milk, and gelatin are widely used.

[0375] G. Immunoassay Labels

[0376] The labeling agent can be, e.g., a monoclonal antibody, apolyclonal antibody, a binding protein or complex, or a polymer such asan affinity matrix, carbohydrate or lipid. Detectable labels suitablefor use in the present invention include any composition detectable byspectroscopic, radioisotopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Detection mayproceed by any known method, such as immunoblotting, western analysis,gel-mobility shift assays, fluorescent in situ hybridization analysis(FISH), tracking of radioactive or bioluminescent markers, nuclearmagnetic resonance, electron paramagnetic resonance, stopped-flowspectroscopy, column chromatography, capillary electrophoresis, or othermethods which track a molecule based upon an alteration in size and/orcharge. The particular label or detectable group used in the assay isnot a critical aspect of the invention. The detectable group can be anymaterial having a detectable physical or chemical property. Suchdetectable labels have been well-developed in the field of immunoassaysand, in general, any label useful in such methods can be applied to thepresent invention. Thus, a label is any composition detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical or chemical means. Useful labels in the present inventioninclude magnetic beads, fluorescent dyes, radiolabels, enzymes, andcalorimetric labels or colored glass or plastic beads, as discussed fornucleic acid labels, supra.

[0377] The label may be coupled directly or indirectly to the desiredcomponent of the assay according to methods well known in the art. Asindicated above, a wide variety of labels may be used, with the choiceof label depending on the sensitivity required, ease of conjugation ofthe compound, stability requirements, available instrumentation, anddisposal provisions.

[0378] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0379] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

[0380] Means of detecting labels are well known to those of skill in theart. Thus, for example, where the label is a radioactive label, meansfor detection include a scintillation counter or photographic film as inautoradiography. Where the label is a fluorescent label, it may bedetected by exciting the fluorochrome with the appropriate wavelength oflight and detecting the resulting fluorescence, e.g., by microscopy,visual inspection, via photographic film, by the use of electronicdetectors such as charge coupled devices (CCDs) or photomultipliers andthe like. Similarly, enzymatic labels may be detected by providingappropriate substrates for the enzyme and detecting the resultingreaction product. Finally, simple calorimetric labels may be detectedsimply by observing the color associated with the label. Thus, invarious dipstick assays, conjugated gold often appears pink, whilevarious conjugated beads appear the color of the bead.

[0381] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0382] Assays for Compounds that Modulate Enzymatic Activity orExpression

[0383] The present invention also provides means for identifyingcompounds that bind to (e.g., substrates), and/or increase or decrease(i.e., modulate) the activity of active polypeptides of the presentinvention. The method comprises contacting a polypeptide of the presentinvention with a compound whose ability to bind to or modulate enzymeactivity is to be determined. The polypeptide employed will have atleast 20%, preferably at least 30% or 40%, more preferably at least 50%or 60%, and most preferably at least 70% or 80% of the specific activityof the native, full-length cell cycle polypeptide (e.g., enzyme).Generally, the polypeptide will be present in a range sufficient todetermine the effect of the compound, typically about 1 nM to 10 μM.Likewise, the compound will be present in a concentration of from about1 nM to 10 μM. Those of skill will understand that such factors asenzyme concentration, ligand concentrations (i.e., substrates, products,inhibitors, activators), pH, ionic strength, and temperature will becontrolled so as to obtain useful kinetic data and determine thepresence of absence of a compound that binds or modulates polypeptideactivity. Methods of measuring enzyme kinetics are well known in theart. See, e.g., Segel, Biochemical Calculations, 2^(nd) ed., John Wileyand Sons, New York (1976).

[0384] Although the present invention has been described in some detailby way of illustration and example for purposes of clarity ofunderstanding, it Will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

[0385] Clones of ZmCycDa-1 and ZmCycDc-1 are on deposit with theAmerican Type Culture Collection (ATCC). The ATCC is at 10801 UniversityBoulevard, Manassas, Va. 20110-2209. The deposits have been made underthe terms of the Budapest Treaty and given the ATCC designation 98848and 98847 respectively.

[0386] During the pendency of this patent application, access to thedeposited cultures will be available to the Commissioner of Patents andTrademarks and to persons determined by the Commissioner to be entitledthereto under 37 CFR 1.14 and 35 U.S.C. 122.

[0387] All restrictions imposed by the depositor on the availability tothe public of the deposited material will be irrevocably removed uponthe granting of a patent. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentaction.

EXAMPLES Example 1

[0388] Isolation of Maize CycD Genes

[0389] Total RNA was isolated from corn tissues with TRIzol Reagent(Life Technology Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi [Chomczynski, P., and Sacchi, N., Anal. Biochem. 162, 156(1987)]. In brief, plant tissue samples were pulverized in liquidnitrogen before the addition of the TRIzol Reagent, and then werefurther homogenized with a mortar and pestle. Addition of chloroformfollowed by centrifugation was conducted for separation of an aqueousphase and an organic phase. The total RNA was recovered by precipitationwith isopropyl alcohol from the aqueous phase.

[0390] Poly(A)+ RNA Isolation:

[0391] The selection of poly(A)+ RNA from total RNA was performed usingPolyATract system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed using high stringency conditions and eluted using RNase-freedeionized water.

[0392] cDNA Library Construction:

[0393] cDNA synthesis was performed and unidirectional cDNA librarieswere constructed using the SuperScript Plasmid System (Life TechnologyInc. Gaithersburg, Md.). The first stand of cDNA was synthesized bypriming an oligo(dT) primer containing a Not I site. The reaction wascatalyzed by SuperScript Reverse Transcriptase II at 45° C. The secondstrand of cDNA was labeled with alpha-³²P-dCTP and a portion of thereaction was analyzed by agarose gel electrophoresis to determine cDNAsizes. cDNA molecules smaller than 500 base pairs and unligated adapterswere removed by Sephacryl-S400 chromatography. The selected cDNAmolecules were ligated into pSPORT1 vector in between Not I and Sal Isites. Mitotically active tissues from Zea mays were employed, includingsuch sources as shoot cultures, immature inflorescences (tassel and ear)as well as other sources of vegetative meristems.

[0394] Sequencing Template Preparation:

[0395] Individual colonies were picked and DNA was prepared either byPCR with M13 forward primers and M13 reverse primers, or by plasmidisolation. All the cDNA clones were initially sequenced using M13reverse primers. As additional fragments of the genes were discovered,new sequencing primers were designed.

[0396]PROTOCOLS, Murray (ed.), pages 271-281 (Humana Press, Inc. 1991).Functional fragments of the cell cycle protein are identified by theirability, upon introduction to cells, to stimulate the G1 to S-phasetransition, which is manifested by increased DNA replication in apopulation of cells and by increased cell division rates.

[0397] 5′-RACE

[0398] Library RACE was performed using several of Pioneer's maizelibraries. 5′ RACE was done using a cDNA library constructed from leavesand stems of maize plants at the three-leaf stage. The principal of 5′RACE is described in detail in numerous publications such as: Frohman M.A. 1993. Rapid Amplification of Complementary DNA Ends for Generation ofFull-Length Complementary DNAs: Thermal RACE. In: Methods in Enzymology,vol. 28, pp 340-356. Detailed procedure can be found in the ClonTechMarathon cloning manual.

Example 2

[0399] Using CycD's in a Two-Hybrid System to Identify Maize Cell CycleGenes

[0400] CycD gene expression during the G1→S transition and early S-phaseplay a prominent role in progression through the cell cycle. Theproteins encoded by the CycD gene family are a critical part of thecomplex that binds and phosphorylates retinoblastoma-associated genefamily members. In turn, Rb releases E2F and this transcription factorstarts the cascade of events leading to DNA replication. As such, theCycD genes and their encoded proteins can be used to identify other cellcycle regulatory proteins. This can be done using the CycD gene as bait(the target fused to the DNA-binding domain) in a yeast two-hybridscreen. Methods for two-hybrid library construction, cloning of thereporter gene, cloning of the DNA-binding and activation domain hybridgene cassettes, yeast culture, and transformation of the yeast are alldone according to well-established methods (see Sambrook et al., 1990;Ausubel et al., 1990; Hannon and Bartels, 1995). Using this method, Zeamays Cdc2, Cdk4 and Rb genes are:identified as components of theactivation domain hybrid, and are confirmed through further sequenceanalysis. Similarly, inhibitors of the Cdk4/CycD complex such as CIP andInk are identified.

Example 3

[0401] CycD-Bound Affinity Columns for Identifying Cdk4 Proteins andtheir Encoding Genes

[0402] Purified recombinant CycD protein can be immobilized on a matrixvia a covalent crosslinking or affinity purification as described supra.This matrix can then be used to pull-down proteins that interact withCycD proteins, inter alia, cyclin-dependent kinase. CDK activity canthen be assessed by measuring the addition of radioactive phosphorus toprotein-substrates and CDK protein levels determined by immunoassay.Additionally, this can be used to purify the CDK activity present indifferent plant tissues and protein fractions. The presence and level ofother CycD interacting proteins can also be determined on the basis ofimmunological assay, activity quantification, SDS-PAGE analysis andother methods. These measures can then be correlated with thereproductive state, capacity for division, developmental stage, or thequality of different samples. A CycD nucleic acid can also be adductedto a second nucleic acid sequence encoding a DNA-binding domain in orderto identify CycD interacting proteins.

Example 4

[0403] Transient CycD Expression Stimulates DNA Replication and EnhancesTransgene Integration

[0404] Regardless of the method of DNA delivery, cells competent for theintegration of foreign DNA must be actively dividing. There is a growingbody of evidence suggesting that integration of foreign DNA occurs individing cells (this includes both Agrobacterium and direct DNA deliverymethods). It has long been observed that dividing transformed cellsrepresent only a fraction of cells that transiently express a transgene.It is well known (in non-plant systems) that the delivery of damagedDNA, (similar to what we introduce by particle gun delivery methods)induces an immediate cell cycle arrest, a process involving cyclindependent kinase inhibitors (CDKI's). This inhibition can be obviated byectopic transient over-expression of positive cell cycle regulators orby down-regulation of negative regulators. Regardless of the mechanismof arrest; i.e. presence of damaged DNA or delivery into a non-cyclingdifferentiated cell, stimulation of the cell cycle will increaseintegration frequencies. To demonstrate this, the CycD gene is clonedinto a cassette with a constitutive promoter (i.e. either a strong maizepromoter such as the ubiquitin promoter including the first ubiquitinintron, or a weak constitutive promoter such as nos). Delivery of theZmCycD gene in an appropriate plant expression cassette (for example, ina UBI::ZmCycD::pinII-containing plasmid) along with UBI::bar::pinII canbe accomplished through numerous well-established methods for plantcells, including for example particle bombardment, sonication, PEGtreatment or electroporation of protoplasts, electroporation of intacttissue, silica-fiber methods, microinjection or Agrobacterium-mediatedtransformation. Using one of the above methods, DNA is introduced intomaize cells capable of growth on suitable maize culture medium. Suchcompetent cells can be from maize suspension culture, callus culture onsolid medium, freshly isolated immature embryos or meristem cells.Immature embryos of the Hi-II genotype are used as the target forco-delivery of these two plasmids. Transient expression of the CycD geneovercomes the G1/S checkpoint controls, and increases the proportion ofrecipient-cells (i.e. into which DNA was introduced) that enter S-phase.This stimulation through the G1/S transition in cells harboringtransgenic plasmid DNA provides an optimal cellular environment forintegration of the introduced genes. Cytological methods can be used toverify increased frequencies of progression through S-phase and mitosis(i.e. for cells in which a visual marker such as GFP was transformedalongside CycD the green fluorescent cells will exhibit a higher mitoticindex). Cells in S-phase (undergoing DNA replication) can be monitoredby detecting nucleotide analog incorporation. For example, followingincubation of cells with bromodeoxyuridine (BrdU) incorporation of thisthymadine analog can be detected by methods such as antiBrdUimmunocytochemistry or through enhancement of Topro3 fluorescencefollowing BrdU labeling. It is expected that CycD expression willincrease the proportion of cells incorporating BrdU (i.e. a higherpercentage of transformed cells will incorporate BrdU relative tountransformed cells). Increased DNA synthesis can also be monitoredusing such methods as fluorescence activated cell sorting (FACS) ofprotoplasts (or nuclei), in conjunction with appropriateBrdU-insensitive fluorescent DNA labels such as propidium iodide andDAPI or BrdU-detecting methods described above. For example, tissue ishomogenized to release nuclei that are analyzed using the FACS for bothgreen fluorescence (from our accompanying GFP marker) and DNA content.Such FACS analysis can demonstrate that expression of a co-transformedGFP reporter correlates with CycD-induced changes in the ratios of cellsin G1, S and G2. Similar experiments can be run using the fluorescentlylabeled anti-BrdU antisera to demonstrate that CycD expression increasedthe percentage of cells in S-phase. Cell cycle stage-specific probes canalso be used to monitor cell cycle progression. For example, numerousspindle-associated proteins are expressed during a fairly narrow windowduring mitosis, and antibodies or nucleic acid probes to cyclins,histones, or DNA synthesis enzymes can be used as positive markers forthe G1/S transition. For cells that have received the CycD genecassette, stimulation of the cell cycle is manifested in an increasedmitotic index, detected by staining for mitotic figures using a DNA dyesuch as DAPI or Hoechst 33258. FACS analysis of CycD-expressing cells isexpected to show that a high percentage of cells have progressed into orthrough S-phase. Progression through S-phase will be manifested by fewercells in G1 and/or more rapid cycling times (i.e. shorter G1 and G2stages). A higher percentage of cells are labeled when cell cyclestage-specific probes are used, as mentioned above.

[0405] To assess the effect on transgene integration, growth ofbialaphos-resistant colonies on selective medium is a reliable assay.Within 1-7 days after DNA introduction, the embryos are moved ontoculture medium containing 3 mg/l of the selective agent bialaphos.Embryos, and later callus, are transferred to fresh selection platesevery 2 weeks. After 6-8 weeks, transformed calli are recovered.Transgenic callus containing the introduced genes can be verified usingPCR and Southern analysis. Northern analysis can also be used to verifywhich calli are expressing the bar gene, and whether the CycD gene isbeing expressed at levels above normal wild-type cells (based onhybridization of probes to freshly isolated mRNA population from thecells). In immature embryos that had transient, elevated CycDexpression, higher numbers of stable transformants are recovered (likelya direct result of increased integration frequencies). Increasedtransgene integration frequency can also be assessed using suchwell-established labeling methods such as in situ hybridization.

[0406] For this specific application (using transient CycD-mediated cellcycle stimulation to increase transient integration frequencies), it maybe desirable to reduce the likelihood of ectopic stable expression ofthe CycD gene. Strategies for transient-only expression can be used.This includes delivery of RNA (transcribed from the CycD gene) or CycDprotein along with the transgene cassettes to be integrated to enhancetransgene integration by transient stimulation of cell division. Usingwell-established methods to produce CycD-RNA, this can then be purifiedand introduced into maize cells using physical methods such asmicroinjection, bombardment, electroporation or silica fiber methods.For protein delivery, the gene is first expressed in a bacterial orbaculoviral system, the protein purified and then introduced into maizecells using physical methods such as microinjection, bombardment,electroporation or silica fiber methods. Alternatively, CycD proteinsare delivered from Agrobacterium tumefaciens into plant cells in theform of fusions to Agrobacterium virulence proteins. Fusions areconstructed between CycD and bacterial virulence proteins such as VirE2,VirD2, or VirF which are known to be delivered directly into plantcells. Fusions are constructed to retain both those properties ofbacterial virulence proteins required to mediate delivery into plantcells and the CycD activity required for enhancing transgeneintegration. This method should ensure a high frequency of simultaneousco-delivery of T-DNA and functional CycD protein into the same hostcell. The methods above represent various means of using the CycD geneor its encoded product to transiently stimulate DNA replication and celldivision, which in turn enhances transgene integration by providing animproved cellular/molecular environment for this) event to occur.

Example 5

[0407] Altering CycD Expression Stimulated the Cell Cycle, IncreasingIntegration and Growth

[0408] Based on results in other eukaryotes, expression of ZmCycD genesstimulates the G1/S transition and promotes cell division. This increasein division rate is assessed in a number of different manners, morerapid incorporation of radiolabeled nucleotides, and faster growth (i.e.more biomass accumulation). Delivery of the ZmCycD in an appropriateplant expression cassette is accomplished through numerouswell-established methods for plant cells, including for example particlebombardment, sonication, PEG treatment or electroporation ofprotoplasts, electroporation of intact tissue, silica-fiber methods,microinjection or Agrobacterium-mediated transformation. The result ofZmCycD gene expression will be to stimulate the G1/S transition andhence cell division, providing the optimal cellular environment forintegration of introduced genes (as per Example 1). This will trigger atissue culture response (cell divisions) in genotypes that typically donot respond to conventional culture techniques, or stimulate growth oftransgenic tissue beyond the normal rates observed in wild-type(non-transgenic) tissues. To demonstrate this, the CycD gene (ZmCycDc-1)was cloned into a cassette with a constitutive promoter (the ubiquitinpromoter, UBI, including the first ubiquitin intron). Particlebombardment was used to introduce the UBI::ZmCycDc-1::pinII-containingplasmid along with a UBI::PAT˜GFP::pinII-containing plasmid (which, whenexpressed produced a functional PAT˜GFP fusion protein which conferedbialaphos resistance and green fluorescence) into maize cells capable ofgrowth on suitable maize culture medium. Such competent cells can befrom maize suspension culture, callus culture on solid medium, freshlyisolated immature embryos or meristem cells. Immature embryos of theHi-II genotype were used as the target for co-delivery of these twoplasmids. Ears were harvested at approximately 10 days post-pollination,and 1.2-1.5 mm immature embryos were isolated from the kernels. Theimmature embryos were bombarded from 18-72 hours later. Typically, theimmature embryos were placed on a high-osmoticum medium for 6-18 hoursprior to bombardment, and were left on this medium for an additional 18hours after bombardment.

[0409] DNA Particle Bombardment

[0410] Between 6 and 18 hours prior to bombardment, the immature embryoswere placed on medium with additional osmoticum (MS basal medium,Musashige and Skoog, 1962, Physiol. Plant 15:473-497, with 0.25 Msorbitol). The embryos on the high-osmotic medium were used as thebombardment target.

[0411] For particle bombardment, plasmid DNA (described above) wasprecipitated onto 1.8 μm tungsten particles using standardCaCl₂-spermidine chemistry (see, for example, Klein et al., 1987, Nature327:70-73). Each plate was bombarded once at 600 PSI, using a DuPontHelium Gun (Lowe et al., 1995, Bio/Technol 13:677-682). For typicalmedia formulations used for maize immature embryo isolation, callusinitiation, callus proliferation and regeneration of plants, seeArmstrong, C. 1994. In “The Maize Handbook”, M. Freeling and V. Walbot,eds. Springer Verlag, NY, pp 663-671.

[0412] Selection

[0413] Within 1-7 days the embryos were moved onto N6-based culturemedium containing 3 mg/l of the selective agent bialaphos. Embryos, andlater callus, were transferred to fresh selection plates every 2 weeks.After the first 14 days post-bombardment, the calli developing from theimmature embryos were screened for GFP expression using anepifluorescent dissecting-microscope. Typically, (i.e. in the absence ofa cell cycle gene) this is too early to observe growing multicellulartransformants. Instead, as typical after such a short post-bombardmentduration, numerous GFP-expressing single-cells were observed on controlembryos (where the UBI::PAT˜GFP::pinII plasmid was introduced alone),but GFP-expressing multicellular clusters were not observed. In markedcontrast to the control treatment, when UBI::CycDc-1 was included alongwith the PAT˜GFP marker, numerous GFP+ multicellular clusters wereobserved growing from the immature embryos at this same earlytime-point. This early stimulation and higher number of growingtransformants observed in the CycD treatment, suggest that expression ofthis cell cycle gene increased integration frequencies (thus highernumbers) and stimulated growth of these small colonies after integrationhad occurred (thus, the transformants were clearly visible at this earlyjuncture). After 6-8 weeks, transformed calli were recovered. Intreatments where both the PAT˜GFP gene and CycD were transformed intoimmature embryos, a higher number of growing calli were recovered on theselective medium and callus growth was stimulated (relative totreatments with the bar gene alone). In the first comparativeexperiments of this type, immature embryos were harvested from 30 ears(over a period of 3 months). From each ear, 25 embryos were used for thecontrol and 25 embryos were used for the UBI::CycD treatment. Thus thetotal number of embryos used per treatment was 750. The transformationfrequency (the number of transgene-expressing independent calli relativeto the starting number of embryos) for the control treatment was 2.4%.for the UBI::CycDc-1 treated embryos, the transformation frequency hadincreased to 7.2%.

[0414] A second experiment demonstrated that both the maize CycDa-1 andCycDc-1 genes result in increased transformation frequencies relative tothe control treatment (where the cyclin gene was not included). For thisbombardment experiment (performed in a similar manner to that describedabove), 3 Hi-II ears were harvested at 10 DAP, and the immature embryoswere divided evenly between the 3 treatments (125 embryos pertreatment). Again, transformants appeared at earlier timepoints in thetwo CycD treatments and the final number of transformants in the CycDtreatments was substantially higher (see FIG. 1). When screened for GFPexpression 46 days post-bombardment, no GFP-expressing multicellularcalli were observed in the control treatment, while in the CycDc-1 andCycDa-1 treatments there were macroscopic GFP+ calli at frequencies of0.7 and 2.3%, respectively. After 77 days, the overall transformationfrequency for the control was 7.4%, while for CycDc-1 and CycDa-1 thefrequency had increased to 12.0 and 18.3% respectively. In addition, thecalli in the CycD treatments were substantially larger than in thecontrol treatment, indicating that these genes stimulated growth rates.

[0415] Differences in cell cycle profiles were also observed inCycD-expressing cells relative to control (wild-type) cells. Todemonstrate that overexpression of CycD genes could accelerate celldivision, the cell cycle profile of maize calli expressing Ubi::CycDwere analyzed using a cell sorter (flow cytometry assay). Flow cytometryis a standard method to study cell cycle, using procedures that are wellestablished in the literature, as, for example, in Sonea I M et al., AmJ Vet Res. 1999 60(3):346-53. Briefly, by counting the number of cellsthat are in G1 phase versus the number of cells that are in G2 phase,one can estimate, in a given population, the percentage of cells thatare undergoing cell division. The higher the percentage of cells in G1phase, the less the number of cells that are dividing. Under standardculture conditions, approximately 70% of the G1/G2 cells of maize calliare in the G1 phase. In maize calli expressing CycD genes, alterationsof the distribution of cells in the G1 and G2 phases were observed. In14 out of 19 CycDa-1 expressing events, the proportion of cells in G1phase decreased to below 60%, and in some cases dropped below 30%. Thus,in these 14 CycDa-1 events, more cells were undergoing cell divisioncompared to wild type maize calli. Using a different CycD gene alsoaltered the cell cycle of transformants, but not in as many events.Compared to the 14 out of 19 CycDa-1 expressing events with increasedcell division rates, only two out of 32 CycDc-1 expressing events showedthat the percentage of G1 cells was lower than 60%. In control calliexpressing similar vector genes but lacking a CycD gene, the cell cycleprofile remained similar to that of the non-treated wild type maizecalli.

[0416] Calli from both the CycDa-1 and CycDc-1 treatment regeneratedeasily. Healthy, fertile transgenic plants were grown in the greenhouse.Seed-set on CycD transgenic plants was similar to control plants, andtransgenic progeny were recovered.

[0417] For a given CycD gene, it was also observed that higherexpression levels improved transformation. For this bombardmentexperiment (performed in a similar manner to that described above), 3Hi-II ears were harvested at 10 DAP, and the immature embryos weredivided evenly between the 3 treatments (125 embryos per treatment). Thetreatments included a no-cyclin control (UBI::PAT˜GFP::pinII), or theUBI::PAT˜GFP::pinII marker plus one of three cyclin-expressing plasmids(UBI::CycDc-1, nos::CycDc-1 or UBI::Da-1). For the CycDc-1 gene, thisexperiment compared high levels of cyclin expression (UBI) to low levels(nos). As seen in FIG. 2, the transformation frequency in the controltreatment was 3.0%. When expression was driven by the UBI promoter, thetransformation frequencies for the CycDa-1 and CycDc-1 genes were 14.4and 17.6%, respectively. However, placing the CycDc-1 gene behind thenos promoter resulted in a transformation similar to the control (1.6%).Based on this result, it appears that higher expression levels result incorrespondingly higher recovery of transformants.

Example 6

[0418] Identifying Transformants in the Absence of Chemical Selection

[0419] When the CycD gene is introduced without any additional selectivemarker, transgenic calli can be identified by their ability to grow morerapidly than surrounding wild-type (non-transformed) tissues. Transgeniccallus can be verified using PCR and Southern analysis. Northernanalysis can also be used to verify which calli are expressing the bargene, and which are expressing the maize CycD gene at levels abovenormal wild-type cells (based on hybridization of probes to freshlyisolated mRNA population from the cells).

[0420] Inducible Expression:

[0421] The CycD gene can also be cloned into a cassette with aninducible promoter such as the benzenesulfonamide-inducible promoter.The expression vector is co-introduced into plant cells and afterselection on bialaphos, the transformed cells are exposed to the safener(inducer). This chemical induction of CycD expression should result instimulated G1/S transition and more rapid cell division. The cells arescreened for the presence of ZmCycD RNA by northern, or RT-PCR (usingtransgene specific probes/oligo pairs), for CycD-encoded protein usingCycD-specific antibodies in Westerns or using hybridization. IncreasedDNA replication is detected using BrdU labeling followed by antibodydetection of cells that incorporated this thymidine analogue. Likewise,other cell cycle division assays could be employed, as described above.

Example 7

[0422] Control of CycD Gene Expression using Tissue-Specific orCell-Specific Promoters Provides a Differential Growth Advantage

[0423] CycD gene expression using tissue-specific or cell-specificpromoters stimulates cell cycle progression in the expressing tissues orcells. For example, using a seed-specific promoter will stimulate celldivision rate and result in increased seed biomass. Alternatively,driving CycD expression with a strongly-expressed, early,tassel-specific promoter will enhance development of this entirereproductive structure.

[0424] Expression of CycD genes in other cell types and/or at differentstages of development will similarly stimulate cell division rates.Similar to results observed in Arabidopsis (Doerner et al., 1996),root-specific or root-preferred expression of CycD will result in largerroots and faster growth (i.e. more biomass accumulation).

Example 8

[0425] Meristem Transformation

[0426] Meristem transformation protocols rely on the transformation ofapical initials or cells that can become apical initials followingreorganization due to injury or selective pressure. The progenitors ofthese apical initials differentiate to form the tissues and organs ofthe mature plant (i.e. leaves, stems, ears, tassels, etc.). Themeristems of most angiosperms are layered with each layer having its ownset of initials. Normally in the shoot apex these layers rarely mix. Inmaize the outer layer of the apical meristem, the L1, differentiates toform the epidermis while descendents of cells in the inner layer, theL2, give rise to internal plant parts including the gametes. Theinitials in each of these layers are defined solely by position and canbe replaced by adjacent cells if they are killed or compromised.Meristem transformation frequently targets a subset of the population ofapical initials and the resulting plants are chimeric. If for example, 1of 4 initials in the L1 layer of the meristem are transformed only ¼ ofepidermis would be transformed. Selective pressure can be used toenlarge sectors but this selection must be non-lethal since large groupsof cells are required for meristem function and survival. Transformationof an apical initial with a Cyclin D expression cassette under theexpression of a promoter active in the apical meristem (either meristemspecific or constitutive) would allow the transformed cells to growfaster and displace wildtype initials driving the meristem towardshomogeneity and minimizing the chimeric nature of the plant body. Todemonstrate this, the CycD gene is cloned into a cassette with apromoter that is active within the meristem (i.e. either a strongconstitutive maize promoter such as the ubiquitin promoter including thefirst ubiquitin intron, or a promoter active in meristematic cells suchas the maize histone, cdc2 or actin promoter). Coleoptilar stage embryosare isolated and plated meristem up on a high sucrose maturation medium(see Lowe et al., 1997). The cyclin D expression cassette along with areporter construct such as Ubi:GUS:pinII can then be co-delivered(preferably 24 hours after isolation) into the exposed apical dome usingconventional particle gun transformation protocols. As a control theCycD construct can be replaced with an equivalent amount of pUC plasmidDNA. After a week to 10 days of culture on maturation medium the embryoscan be transferred to a low sucrose hormone-free germination medium.Leaves from developing plants can be sacrificed for GUS staining.Transient expression of the CycD gene in meristem cells, throughstimulation of the G1→S transition, will result in greater integrationfrequencies and hence more numerous transgenic sectors. Integration andexpression of the CycD gene will impart a competitive advantage toexpressing cells resulting in a progressive enlargement of thetransgenic sector. Due to the enhanced growth rate in CycD-expressingmeristem cells, they will supplant wild-type meristem cells as the.plant continues to grow. The result will be both enlargement oftransgenic sectors within a given cell layer (i.e. periclinal expansion)and into adjacent cell layers (i.e. anticlinal invasions). As anincreasingly large proportion of the meristem is occupied byCycD-expressing cells, the frequency of CycD germline inheritance shouldgo up accordingly.

Example 9

[0427] Use of Flp/Frt System to Excise the CycD Cassette

[0428] In cases where the CycD gene has been integrated and CycDexpression is useful in the recovery of maize trangenics, but isultimately not desired in the final product, the CycD expressioncassette (or any portion thereof that is flanked by appropriate FRTrecombination sequences) can be excised using FLP-mediated recombination(see U.S. patent application Ser. No. 08/972,258 filed Nov. 18, 1997).

[0429] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, and patent applicationscited herein are hereby incorporated by reference.

1 30 1 1861 DNA Zea mays CDS (275)...(1351) 1 tcctctgtcc tcccctctccacttgagaag aacacaatta gaaaaaaagg caaaaaacat 60 ttaccttttt tctatctgtatattatctga ataaatcaag aggaggaaga ggggagggag 120 cgagggaggg ggaggagtagcaaatccaga ctccatagca accagctcgc gagaagggga 180 aaagggggag gaagagcttcgcttgtgtat tgattgctcg ctgctccagt ccctgcattc 240 gtgccgtttt tggcaagtaggtggcgtggc aagc atg gtg ccg ggc tat gac tgc 295 Met Val Pro Gly Tyr AspCys 1 5 gcc gcc tcc gtg ctg ctg tgc gcg gag gac aac gct gct att ctc ggc343 Ala Ala Ser Val Leu Leu Cys Ala Glu Asp Asn Ala Ala Ile Leu Gly 1015 20 ctg gac gac gat ggg gag gag tcc tcc tgg gcg gcc gcc gct acg ccg391 Leu Asp Asp Asp Gly Glu Glu Ser Ser Trp Ala Ala Ala Ala Thr Pro 2530 35 cca cgt gac acc gtc gcc gcc gcc gcc gcc acc ggg gtc gcc gtc gat439 Pro Arg Asp Thr Val Ala Ala Ala Ala Ala Thr Gly Val Ala Val Asp 4045 50 55 ggg att ttg acg gag ttc ccc ttg ctc tcg gat gac tgc gtt gcg acg487 Gly Ile Leu Thr Glu Phe Pro Leu Leu Ser Asp Asp Cys Val Ala Thr 6065 70 ctc gtg gag aag gag gtg gag cac atg ccc gcg gag ggg tac ctc cag535 Leu Val Glu Lys Glu Val Glu His Met Pro Ala Glu Gly Tyr Leu Gln 7580 85 aag ctg cag cga cgg cat ggg gac ctg gat ttg gcc gcc gtc agg aag583 Lys Leu Gln Arg Arg His Gly Asp Leu Asp Leu Ala Ala Val Arg Lys 9095 100 gac gcc atc gat tgg att tgg aag gtc att gag cat tac aat ttc gca631 Asp Ala Ile Asp Trp Ile Trp Lys Val Ile Glu His Tyr Asn Phe Ala 105110 115 ccg ttg act gcc gtt ttg tct gtg aac tac ctc gat aga ttc ctc tcc679 Pro Leu Thr Ala Val Leu Ser Val Asn Tyr Leu Asp Arg Phe Leu Ser 120125 130 135 acg tat gag ttc cct gaa ggc aga gct tgg atg act cag ctc ttggca 727 Thr Tyr Glu Phe Pro Glu Gly Arg Ala Trp Met Thr Gln Leu Leu Ala140 145 150 gtg gct tgc ttg tct ttg gct tcg aaa atc gaa gag act ttt gtgcca 775 Val Ala Cys Leu Ser Leu Ala Ser Lys Ile Glu Glu Thr Phe Val Pro155 160 165 ctc ccc ttg gat ttg cag gta gcg gag gca aag ttt gtt ttt gaggga 823 Leu Pro Leu Asp Leu Gln Val Ala Glu Ala Lys Phe Val Phe Glu Gly170 175 180 agg acc ata aaa agg atg gag ctt ctg gtg cta agc acc tta aagtgg 871 Arg Thr Ile Lys Arg Met Glu Leu Leu Val Leu Ser Thr Leu Lys Trp185 190 195 agg atg cat gct gtt act gct tgc tca ttt gtt gaa tac ttt cttcat 919 Arg Met His Ala Val Thr Ala Cys Ser Phe Val Glu Tyr Phe Leu His200 205 210 215 aaa ttg agt gat cat ggt gca ccc tcc ttg ctt gca cgc tctcgc tct 967 Lys Leu Ser Asp His Gly Ala Pro Ser Leu Leu Ala Arg Ser ArgSer 220 225 230 tcg gac ctt gtc ttg agc acc gct aaa ggt gct gaa ttc gtggta ttc 1015 Ser Asp Leu Val Leu Ser Thr Ala Lys Gly Ala Glu Phe Val ValPhe 235 240 245 aga ccc tcc gag att gct gcc agt gtt gca ctt gct gct atcggc gaa 1063 Arg Pro Ser Glu Ile Ala Ala Ser Val Ala Leu Ala Ala Ile GlyGlu 250 255 260 tgc agg agt tct gta att gag aga gct gct agt agc tgc aaatat ttg 1111 Cys Arg Ser Ser Val Ile Glu Arg Ala Ala Ser Ser Cys Lys TyrLeu 265 270 275 gac aag gag agg gtt tta aga tgc cat gaa atg att caa gagaag att 1159 Asp Lys Glu Arg Val Leu Arg Cys His Glu Met Ile Gln Glu LysIle 280 285 290 295 act gcg gga agc att gtc cta aag tct gct gga tca tcaatc tcc tct 1207 Thr Ala Gly Ser Ile Val Leu Lys Ser Ala Gly Ser Ser IleSer Ser 300 305 310 gtg cca caa agc cca ata ggt gtc ctg gac gct gca gcctgt ctg agt 1255 Val Pro Gln Ser Pro Ile Gly Val Leu Asp Ala Ala Ala CysLeu Ser 315 320 325 caa caa agc gat gac gct act gtc ggg tct cct gca gtatgt tac cat 1303 Gln Gln Ser Asp Asp Ala Thr Val Gly Ser Pro Ala Val CysTyr His 330 335 340 agt tct tcc aca agc aag agg aga agg atc act aga cgtcta ctc taa 1351 Ser Ser Ser Thr Ser Lys Arg Arg Arg Ile Thr Arg Arg LeuLeu * 345 350 355 ttgtggtacg cttcaggtgt gctcctcacc gctctaggag tttttgattggttcaaacat 1411 cttaaattta gtttggccgc tggaggatta tggtttagtc aagtagttgctgaatggaac 1471 aacaaaacac gcacactact tggtccataa agacaagaaa ataactggcagcgtcccgcg 1531 agccagcgct gcaatccagt tcatgcaaga ccctagagtc cagggggggtgctggtgtag 1591 gtagagaggg aacaaggcat tcacatacgc cgtagagatg agagagcctctcgtatgttt 1651 tgtacttttg ctccttcagt ttgcaatgaa ctatataaac aaggattgccttggggcagt 1711 gaacatttgt cggatgaaaa gaatcaaaaa ggatgggggt cggcagaggaatagaacaat 1771 ttgatatatt tccataaact acagatatgt ttcctttttc ataatgatgagctatcattt 1831 ttgttgatgg taacaaaaaa aaaaaaaaaa 1861 2 358 PRT Zea mays2 Met Val Pro Gly Tyr Asp Cys Ala Ala Ser Val Leu Leu Cys Ala Glu 1 5 1015 Asp Asn Ala Ala Ile Leu Gly Leu Asp Asp Asp Gly Glu Glu Ser Ser 20 2530 Trp Ala Ala Ala Ala Thr Pro Pro Arg Asp Thr Val Ala Ala Ala Ala 35 4045 Ala Thr Gly Val Ala Val Asp Gly Ile Leu Thr Glu Phe Pro Leu Leu 50 5560 Ser Asp Asp Cys Val Ala Thr Leu Val Glu Lys Glu Val Glu His Met 65 7075 80 Pro Ala Glu Gly Tyr Leu Gln Lys Leu Gln Arg Arg His Gly Asp Leu 8590 95 Asp Leu Ala Ala Val Arg Lys Asp Ala Ile Asp Trp Ile Trp Lys Val100 105 110 Ile Glu His Tyr Asn Phe Ala Pro Leu Thr Ala Val Leu Ser ValAsn 115 120 125 Tyr Leu Asp Arg Phe Leu Ser Thr Tyr Glu Phe Pro Glu GlyArg Ala 130 135 140 Trp Met Thr Gln Leu Leu Ala Val Ala Cys Leu Ser LeuAla Ser Lys 145 150 155 160 Ile Glu Glu Thr Phe Val Pro Leu Pro Leu AspLeu Gln Val Ala Glu 165 170 175 Ala Lys Phe Val Phe Glu Gly Arg Thr IleLys Arg Met Glu Leu Leu 180 185 190 Val Leu Ser Thr Leu Lys Trp Arg MetHis Ala Val Thr Ala Cys Ser 195 200 205 Phe Val Glu Tyr Phe Leu His LysLeu Ser Asp His Gly Ala Pro Ser 210 215 220 Leu Leu Ala Arg Ser Arg SerSer Asp Leu Val Leu Ser Thr Ala Lys 225 230 235 240 Gly Ala Glu Phe ValVal Phe Arg Pro Ser Glu Ile Ala Ala Ser Val 245 250 255 Ala Leu Ala AlaIle Gly Glu Cys Arg Ser Ser Val Ile Glu Arg Ala 260 265 270 Ala Ser SerCys Lys Tyr Leu Asp Lys Glu Arg Val Leu Arg Cys His 275 280 285 Glu MetIle Gln Glu Lys Ile Thr Ala Gly Ser Ile Val Leu Lys Ser 290 295 300 AlaGly Ser Ser Ile Ser Ser Val Pro Gln Ser Pro Ile Gly Val Leu 305 310 315320 Asp Ala Ala Ala Cys Leu Ser Gln Gln Ser Asp Asp Ala Thr Val Gly 325330 335 Ser Pro Ala Val Cys Tyr His Ser Ser Ser Thr Ser Lys Arg Arg Arg340 345 350 Ile Thr Arg Arg Leu Leu 355 3 27 DNA Zea mays protein_bind(1)...(27) 3 gcaagcatgg tgccgggcta tgactgc 27 4 30 DNA Zea maysprotein_bind (1)...(30) 4 agcggtgagg agcacacctg aagcgtacca 30 5 27 DNAZea mays primer_bind (1)...(27) 5 tctattcctc tgccgacccc catcctt 27 6 30DNA Zea mays protein_bind (1)...(30) 6 cccctctcca cttgagaaga acacaattag30 7 25 DNA Zea mays primer_bind (1)...(25) 7 cgggctatga ctgcgccgcctccgt 25 8 27 DNA Zea mays protein_bind (1)...(27) 8 ctcctcttgcttgtggaaga actatgg 27 9 25 DNA Zea mays primer_bind (1)...(25) 9atggtgccgg gctatgactg cgccg 25 10 25 DNA Zea mays protein_bind(1)...(25) 10 ttagagtaga cgtctagtga tcctt 25 11 1077 DNA Zea mays CDS(1)...(1077) 11 atg gtg ccg ggc tat gac tgc gcc gcc tcc gtg ctg ctg tgcgcg gag 48 Met Val Pro Gly Tyr Asp Cys Ala Ala Ser Val Leu Leu Cys AlaGlu 1 5 10 15 gac aac gct gct att ctc ggc ctg gac gac gat ggg gag gagtcc tcc 96 Asp Asn Ala Ala Ile Leu Gly Leu Asp Asp Asp Gly Glu Glu SerSer 20 25 30 tgg gcg gcc gcc gct acg ccg cca cgt gac acc gtc gcc gcc gccgcc 144 Trp Ala Ala Ala Ala Thr Pro Pro Arg Asp Thr Val Ala Ala Ala Ala35 40 45 gcc acc ggg gtc gcc gtc gat ggg att ttg acg gag ttc ccc ttg ctc192 Ala Thr Gly Val Ala Val Asp Gly Ile Leu Thr Glu Phe Pro Leu Leu 5055 60 tcg gat gac tgc gtt gcg acg ctc gtg gag aag gag gtg gag cac atg240 Ser Asp Asp Cys Val Ala Thr Leu Val Glu Lys Glu Val Glu His Met 6570 75 80 ccc gcg gag ggg tac ctc cag aag ctg cag cga cgg cat ggg gac ctg288 Pro Ala Glu Gly Tyr Leu Gln Lys Leu Gln Arg Arg His Gly Asp Leu 8590 95 gat ttg gtc gcc gtc agg aag gac gcc atc gat tgg att tgg aag gtc336 Asp Leu Val Ala Val Arg Lys Asp Ala Ile Asp Trp Ile Trp Lys Val 100105 110 att gag cat tac aat ttc gca ccg ttg act gcc gtt ttg tct gtg aac384 Ile Glu His Tyr Asn Phe Ala Pro Leu Thr Ala Val Leu Ser Val Asn 115120 125 tac ctc gat aga ttc ctc tcc acg tat gag ttc cct gaa ggc aga gct432 Tyr Leu Asp Arg Phe Leu Ser Thr Tyr Glu Phe Pro Glu Gly Arg Ala 130135 140 tgg atg act cag ctc ttg gca gtg gct tgc ttg tct ttg gct tcg aaa480 Trp Met Thr Gln Leu Leu Ala Val Ala Cys Leu Ser Leu Ala Ser Lys 145150 155 160 atc gaa gag act ttt gtg cca ctc ccc ttg gat ttg cag gta gcggag 528 Ile Glu Glu Thr Phe Val Pro Leu Pro Leu Asp Leu Gln Val Ala Glu165 170 175 gca aag ttt gtt ttt gag gga agg acc ata aaa agg atg gag cttctg 576 Ala Lys Phe Val Phe Glu Gly Arg Thr Ile Lys Arg Met Glu Leu Leu180 185 190 gtg cta agc acc tta aag tgg agg atg cat gct gtt act gct tgctca 624 Val Leu Ser Thr Leu Lys Trp Arg Met His Ala Val Thr Ala Cys Ser195 200 205 ttt gtt gaa tac ttt ctt cat aaa ttg agt gat cat ggt gca ccctcc 672 Phe Val Glu Tyr Phe Leu His Lys Leu Ser Asp His Gly Ala Pro Ser210 215 220 ttg ctt gca cgc tct cgc tct tcg gac ctt gtc ttg agc acc gctaaa 720 Leu Leu Ala Arg Ser Arg Ser Ser Asp Leu Val Leu Ser Thr Ala Lys225 230 235 240 ggt gct gaa ttc gtg gta ttc aga ccc tcc gag att gct gccagt gtt 768 Gly Ala Glu Phe Val Val Phe Arg Pro Ser Glu Ile Ala Ala SerVal 245 250 255 gca ctt gct gct atc ggc gaa tgc agg agt tct gta att gagaga gct 816 Ala Leu Ala Ala Ile Gly Glu Cys Arg Ser Ser Val Ile Glu ArgAla 260 265 270 gct agt agc tgc aaa tat ttg gac aag gag agg gtt tta agatgc cat 864 Ala Ser Ser Cys Lys Tyr Leu Asp Lys Glu Arg Val Leu Arg CysHis 275 280 285 gaa atg att caa gag aag att act atg gga agc att gtc ctaaag tct 912 Glu Met Ile Gln Glu Lys Ile Thr Met Gly Ser Ile Val Leu LysSer 290 295 300 gct gga tca tca atc tcc tct gtg cca caa agc cca ata ggtgtc ctg 960 Ala Gly Ser Ser Ile Ser Ser Val Pro Gln Ser Pro Ile Gly ValLeu 305 310 315 320 gac gct gca gcc tgt ctg agt caa caa agc gat gac gctact gtc ggg 1008 Asp Ala Ala Ala Cys Leu Ser Gln Gln Ser Asp Asp Ala ThrVal Gly 325 330 335 tct cct gca gta tgt tac cat agt tct tcc aca agc aagagg aga atg 1056 Ser Pro Ala Val Cys Tyr His Ser Ser Ser Thr Ser Lys ArgArg Met 340 345 350 atc act aga cgt cta ctc taa 1077 Ile Thr Arg Arg LeuLeu * 355 12 358 PRT Zea mays 12 Met Val Pro Gly Tyr Asp Cys Ala Ala SerVal Leu Leu Cys Ala Glu 1 5 10 15 Asp Asn Ala Ala Ile Leu Gly Leu AspAsp Asp Gly Glu Glu Ser Ser 20 25 30 Trp Ala Ala Ala Ala Thr Pro Pro ArgAsp Thr Val Ala Ala Ala Ala 35 40 45 Ala Thr Gly Val Ala Val Asp Gly IleLeu Thr Glu Phe Pro Leu Leu 50 55 60 Ser Asp Asp Cys Val Ala Thr Leu ValGlu Lys Glu Val Glu His Met 65 70 75 80 Pro Ala Glu Gly Tyr Leu Gln LysLeu Gln Arg Arg His Gly Asp Leu 85 90 95 Asp Leu Val Ala Val Arg Lys AspAla Ile Asp Trp Ile Trp Lys Val 100 105 110 Ile Glu His Tyr Asn Phe AlaPro Leu Thr Ala Val Leu Ser Val Asn 115 120 125 Tyr Leu Asp Arg Phe LeuSer Thr Tyr Glu Phe Pro Glu Gly Arg Ala 130 135 140 Trp Met Thr Gln LeuLeu Ala Val Ala Cys Leu Ser Leu Ala Ser Lys 145 150 155 160 Ile Glu GluThr Phe Val Pro Leu Pro Leu Asp Leu Gln Val Ala Glu 165 170 175 Ala LysPhe Val Phe Glu Gly Arg Thr Ile Lys Arg Met Glu Leu Leu 180 185 190 ValLeu Ser Thr Leu Lys Trp Arg Met His Ala Val Thr Ala Cys Ser 195 200 205Phe Val Glu Tyr Phe Leu His Lys Leu Ser Asp His Gly Ala Pro Ser 210 215220 Leu Leu Ala Arg Ser Arg Ser Ser Asp Leu Val Leu Ser Thr Ala Lys 225230 235 240 Gly Ala Glu Phe Val Val Phe Arg Pro Ser Glu Ile Ala Ala SerVal 245 250 255 Ala Leu Ala Ala Ile Gly Glu Cys Arg Ser Ser Val Ile GluArg Ala 260 265 270 Ala Ser Ser Cys Lys Tyr Leu Asp Lys Glu Arg Val LeuArg Cys His 275 280 285 Glu Met Ile Gln Glu Lys Ile Thr Met Gly Ser IleVal Leu Lys Ser 290 295 300 Ala Gly Ser Ser Ile Ser Ser Val Pro Gln SerPro Ile Gly Val Leu 305 310 315 320 Asp Ala Ala Ala Cys Leu Ser Gln GlnSer Asp Asp Ala Thr Val Gly 325 330 335 Ser Pro Ala Val Cys Tyr His SerSer Ser Thr Ser Lys Arg Arg Met 340 345 350 Ile Thr Arg Arg Leu Leu 35513 1173 DNA Zea mays CDS (1)...(1173) 13 atg gcg ccg agc tgc tac gac gcggca gcg tcc atg ctc ctc tgc gcc 48 Met Ala Pro Ser Cys Tyr Asp Ala AlaAla Ser Met Leu Leu Cys Ala 1 5 10 15 gag gag cac agc agc atc ctg tggtac gac gag gag gag gag gag ctg 96 Glu Glu His Ser Ser Ile Leu Trp TyrAsp Glu Glu Glu Glu Glu Leu 20 25 30 gag gcg gtc ggg aga agg aga ggc cggtcg ccg ggc tac ggg gac gac 144 Glu Ala Val Gly Arg Arg Arg Gly Arg SerPro Gly Tyr Gly Asp Asp 35 40 45 ttc ggc gcg gac ttg ttc ccg ccg cag tcggag gaa tgc gtg gcc ggt 192 Phe Gly Ala Asp Leu Phe Pro Pro Gln Ser GluGlu Cys Val Ala Gly 50 55 60 ctg gtg gag cgg gaa cgg gac cac atg ccg gggccg tgc tac ggc gac 240 Leu Val Glu Arg Glu Arg Asp His Met Pro Gly ProCys Tyr Gly Asp 65 70 75 80 agg ctg cgc ggc ggc ggc ggc tgt ctc tgc gtccgc cgg gag gcc gtc 288 Arg Leu Arg Gly Gly Gly Gly Cys Leu Cys Val ArgArg Glu Ala Val 85 90 95 gac tgg att tgg aag gct tac acg cac cac agg ttccgc cct ctc act 336 Asp Trp Ile Trp Lys Ala Tyr Thr His His Arg Phe ArgPro Leu Thr 100 105 110 gcc tac ttg gca gtg aac tac ctc gat cgc ttc ctctcg ctg tct gag 384 Ala Tyr Leu Ala Val Asn Tyr Leu Asp Arg Phe Leu SerLeu Ser Glu 115 120 125 gtg ccg gac ggc aag gac tgg atg acg cag ctc ctcgcg gtg gcg tgc 432 Val Pro Asp Gly Lys Asp Trp Met Thr Gln Leu Leu AlaVal Ala Cys 130 135 140 gtt tct ctg gcc gcc aag atg gag gaa acc gcc gtcccg cag tgc ctg 480 Val Ser Leu Ala Ala Lys Met Glu Glu Thr Ala Val ProGln Cys Leu 145 150 155 160 gac ctt cag gtc gga gac gcg cgg tac gtg ttcgag gcg aag acg gtc 528 Asp Leu Gln Val Gly Asp Ala Arg Tyr Val Phe GluAla Lys Thr Val 165 170 175 cag agg atg gag ctc ctg gtt cta aca acc ctcaac tgg agg atg cat 576 Gln Arg Met Glu Leu Leu Val Leu Thr Thr Leu AsnTrp Arg Met His 180 185 190 gcc gtg acg ccg ttc tcc tac gtg gat tac ttcctg aac aag ctc agc 624 Ala Val Thr Pro Phe Ser Tyr Val Asp Tyr Phe LeuAsn Lys Leu Ser 195 200 205 aac ggc ggc agc acg gcg ccg agg agc tgc tggctc ttg cag tcc gcg 672 Asn Gly Gly Ser Thr Ala Pro Arg Ser Cys Trp LeuLeu Gln Ser Ala 210 215 220 gag ctt atc ttg cgt gcg gcc aga gga acc ggctgc gtc ggg ttc agg 720 Glu Leu Ile Leu Arg Ala Ala Arg Gly Thr Gly CysVal Gly Phe Arg 225 230 235 240 ccg tcc gag atc gcc gcc gcg gtt gca gccgcc gtg gcc gga gac gtg 768 Pro Ser Glu Ile Ala Ala Ala Val Ala Ala AlaVal Ala Gly Asp Val 245 250 255 gac gac gcg gac ggc gtc gag aac gcc tgctgc gct cac gta gat aag 816 Asp Asp Ala Asp Gly Val Glu Asn Ala Cys CysAla His Val Asp Lys 260 265 270 gag cgg gtg ttg cgg tgc cag gaa gcg atcggg tcc atg gcg tcc tcg 864 Glu Arg Val Leu Arg Cys Gln Glu Ala Ile GlySer Met Ala Ser Ser 275 280 285 gcg gcc att gac ggc gac gct acc gtg ccaccg aaa tct gcg aga cgc 912 Ala Ala Ile Asp Gly Asp Ala Thr Val Pro ProLys Ser Ala Arg Arg 290 295 300 agg agc tcc ccc gtg ccc gtg ccc gtg cccgtg ccg cag agc cct gtg 960 Arg Ser Ser Pro Val Pro Val Pro Val Pro ValPro Gln Ser Pro Val 305 310 315 320 ggg gtg ctg gac gcg gcc gcc tgc ctcagc tac agg agc gaa gag gca 1008 Gly Val Leu Asp Ala Ala Ala Cys Leu SerTyr Arg Ser Glu Glu Ala 325 330 335 gcg act gcg act gcg act tct gct gcctca cat ggg ccc cct ggc tct 1056 Ala Thr Ala Thr Ala Thr Ser Ala Ala SerHis Gly Pro Pro Gly Ser 340 345 350 tca agc tcg tcc tcg acc tcc ccg gtgacc agc aaa agg agg aaa ctc 1104 Ser Ser Ser Ser Ser Thr Ser Pro Val ThrSer Lys Arg Arg Lys Leu 355 360 365 gcc agc cga tgt gat gga tcg tgc agtgac cgg tca aag cgc gcg ccc 1152 Ala Ser Arg Cys Asp Gly Ser Cys Ser AspArg Ser Lys Arg Ala Pro 370 375 380 gcc caa tgg acc aaa gag tga 1173 AlaGln Trp Thr Lys Glu * 385 390 14 390 PRT Zea mays 14 Met Ala Pro Ser CysTyr Asp Ala Ala Ala Ser Met Leu Leu Cys Ala 1 5 10 15 Glu Glu His SerSer Ile Leu Trp Tyr Asp Glu Glu Glu Glu Glu Leu 20 25 30 Glu Ala Val GlyArg Arg Arg Gly Arg Ser Pro Gly Tyr Gly Asp Asp 35 40 45 Phe Gly Ala AspLeu Phe Pro Pro Gln Ser Glu Glu Cys Val Ala Gly 50 55 60 Leu Val Glu ArgGlu Arg Asp His Met Pro Gly Pro Cys Tyr Gly Asp 65 70 75 80 Arg Leu ArgGly Gly Gly Gly Cys Leu Cys Val Arg Arg Glu Ala Val 85 90 95 Asp Trp IleTrp Lys Ala Tyr Thr His His Arg Phe Arg Pro Leu Thr 100 105 110 Ala TyrLeu Ala Val Asn Tyr Leu Asp Arg Phe Leu Ser Leu Ser Glu 115 120 125 ValPro Asp Gly Lys Asp Trp Met Thr Gln Leu Leu Ala Val Ala Cys 130 135 140Val Ser Leu Ala Ala Lys Met Glu Glu Thr Ala Val Pro Gln Cys Leu 145 150155 160 Asp Leu Gln Val Gly Asp Ala Arg Tyr Val Phe Glu Ala Lys Thr Val165 170 175 Gln Arg Met Glu Leu Leu Val Leu Thr Thr Leu Asn Trp Arg MetHis 180 185 190 Ala Val Thr Pro Phe Ser Tyr Val Asp Tyr Phe Leu Asn LysLeu Ser 195 200 205 Asn Gly Gly Ser Thr Ala Pro Arg Ser Cys Trp Leu LeuGln Ser Ala 210 215 220 Glu Leu Ile Leu Arg Ala Ala Arg Gly Thr Gly CysVal Gly Phe Arg 225 230 235 240 Pro Ser Glu Ile Ala Ala Ala Val Ala AlaAla Val Ala Gly Asp Val 245 250 255 Asp Asp Ala Asp Gly Val Glu Asn AlaCys Cys Ala His Val Asp Lys 260 265 270 Glu Arg Val Leu Arg Cys Gln GluAla Ile Gly Ser Met Ala Ser Ser 275 280 285 Ala Ala Ile Asp Gly Asp AlaThr Val Pro Pro Lys Ser Ala Arg Arg 290 295 300 Arg Ser Ser Pro Val ProVal Pro Val Pro Val Pro Gln Ser Pro Val 305 310 315 320 Gly Val Leu AspAla Ala Ala Cys Leu Ser Tyr Arg Ser Glu Glu Ala 325 330 335 Ala Thr AlaThr Ala Thr Ser Ala Ala Ser His Gly Pro Pro Gly Ser 340 345 350 Ser SerSer Ser Ser Thr Ser Pro Val Thr Ser Lys Arg Arg Lys Leu 355 360 365 AlaSer Arg Cys Asp Gly Ser Cys Ser Asp Arg Ser Lys Arg Ala Pro 370 375 380Ala Gln Trp Thr Lys Glu 385 390 15 24 DNA Zea mays primer_bind(1)...(24) 15 cacgcgcacc agcccaccgc ccag 24 16 25 DNA Zea maysprotein_bind (1)...(25) 16 tcccatcgga tctcctctag cgccc 25 17 21 DNA Zeamays primer_bind (1)...(21) 17 tcactctttg gtccattggg c 21 18 24 DNA Zeamays primer_bind (1)...(24) 18 tcaattcact cttggtccat tggg 24 19 25 DNAZea mays primer_bind (1)...(25) 19 tgcgccgagg agcacagcag catcc 25 20 25DNA Zea mays protein_bind (1)...(25) 20 gaccggtcac tgcacgatcc atcac 2521 1734 DNA Zea mays CDS (213)...(1262) 21 ccacgcgtcc ggggagggaattccttcctc cttttctgtt cggcgccgtg ctcgcgcgca 60 cccacccgca cgccccagtacccccacgct gcacagtgca cgccgacttt cctccgcctt 120 gctgctgcaa gtccgcaaccactggaggaa aaatcttttc cttcactttt cttccctttc 180 cccccgcgca tgcacgggctctgattgacg cc atg ggg gac gcc gcg gcc tcc 233 Met Gly Asp Ala Ala AlaSer 1 5 acg tcc gct ccc acc acg ccc acc tcc atc ctc atc tgc ctg gaa gac281 Thr Ser Ala Pro Thr Thr Pro Thr Ser Ile Leu Ile Cys Leu Glu Asp 1015 20 ggc agc gac ctt ctc gcc gat gcc gac gat ggc gcc ggc act gac ctc329 Gly Ser Asp Leu Leu Ala Asp Ala Asp Asp Gly Ala Gly Thr Asp Leu 2530 35 gtt gtc gcc cgc gac gaa cgt ctg ctt gtc gtg gac cag gac gag gag377 Val Val Ala Arg Asp Glu Arg Leu Leu Val Val Asp Gln Asp Glu Glu 4045 50 55 tat gta gcg ctg ctc ctg tcc aag gag agc gcg tca ggc ggc ggc ggc425 Tyr Val Ala Leu Leu Leu Ser Lys Glu Ser Ala Ser Gly Gly Gly Gly 6065 70 ccg gtg gag gaa atg gag gac tgg atg aag gcc gcg cgc tcc gga tgc473 Pro Val Glu Glu Met Glu Asp Trp Met Lys Ala Ala Arg Ser Gly Cys 7580 85 gtc cgc tgg atc atc aag acc acg gcg atg ttc cgg ttc ggc ggg aag521 Val Arg Trp Ile Ile Lys Thr Thr Ala Met Phe Arg Phe Gly Gly Lys 9095 100 acc gct tac gtc gcg gtg aat tac ctc gat cgc ttc ctg gcg caa cgg569 Thr Ala Tyr Val Ala Val Asn Tyr Leu Asp Arg Phe Leu Ala Gln Arg 105110 115 cga gtc aat agg gag cat gcg tgg ggt ctg cag ctg ctc atg gtg gcg617 Arg Val Asn Arg Glu His Ala Trp Gly Leu Gln Leu Leu Met Val Ala 120125 130 135 tgc atg tcg ctg gcg acc aag ctg gag gag cac cac gct ccg cggctg 665 Cys Met Ser Leu Ala Thr Lys Leu Glu Glu His His Ala Pro Arg Leu140 145 150 tcg gag ttg ccc ctg gac gcg tgc gag ttc gcg ttc gac cgc gcgtcc 713 Ser Glu Leu Pro Leu Asp Ala Cys Glu Phe Ala Phe Asp Arg Ala Ser155 160 165 gtg ctg cgg atg gag ctc ctc gtc ctg ggc acc ctc gag tgg cggatg 761 Val Leu Arg Met Glu Leu Leu Val Leu Gly Thr Leu Glu Trp Arg Met170 175 180 gtc gcc gtc acc ccc ttc ccc tac atc agc tgc ttc gcg gcg cggttc 809 Val Ala Val Thr Pro Phe Pro Tyr Ile Ser Cys Phe Ala Ala Arg Phe185 190 195 cgg cag gac gag cgc cgg gcg gtc ctc gtg cgc gcc gtg gag tgcgtc 857 Arg Gln Asp Glu Arg Arg Ala Val Leu Val Arg Ala Val Glu Cys Val200 205 210 215 ttc gcg gcg atc aga gcg atg agc tcg gtg gag tac cag ccgtcg acc 905 Phe Ala Ala Ile Arg Ala Met Ser Ser Val Glu Tyr Gln Pro SerThr 220 225 230 atc gcc gta gca tcc atc ctc gtc gct cgc ggc agg gag acgccc gcc 953 Ile Ala Val Ala Ser Ile Leu Val Ala Arg Gly Arg Glu Thr ProAla 235 240 245 ggc aat ctg gac gcg ctc aag gcg atc ctg ggc tca tca tttccg cag 1001 Gly Asn Leu Asp Ala Leu Lys Ala Ile Leu Gly Ser Ser Phe ProGln 250 255 260 cta gac acc ggg cat gtg tac tcc tgc tac agc gca atg attcgg gag 1049 Leu Asp Thr Gly His Val Tyr Ser Cys Tyr Ser Ala Met Ile ArgGlu 265 270 275 gac gac aag tcg ccg acg cag tcg acg tcg acg tcg acg ggggtg gcg 1097 Asp Asp Lys Ser Pro Thr Gln Ser Thr Ser Thr Ser Thr Gly ValAla 280 285 290 295 tcc tcg ggc gtc tct gtc gcc gcg cac gcc ggg ggg agcggg agt ccc 1145 Ser Ser Gly Val Ser Val Ala Ala His Ala Gly Gly Ser GlySer Pro 300 305 310 agc ccc ccg ggc gct tcc gtg tcc gtg ggc gcc aat aatgcc gct ggc 1193 Ser Pro Pro Gly Ala Ser Val Ser Val Gly Ala Asn Asn AlaAla Gly 315 320 325 acc gcc ccg ccg gca acc acg gac aac cgc aac aag aggaga cgg ttg 1241 Thr Ala Pro Pro Ala Thr Thr Asp Asn Arg Asn Lys Arg ArgArg Leu 330 335 340 cgc tca cct cag cga cag tag gagcagctca gctgctggcagtgcattgca 1292 Arg Ser Pro Gln Arg Gln * 345 gtgcagtgca gtccagctgcgttttctttt ttcagctcac catttccttt tgctgccgat 1352 tgtttcttca ggggtggccgtagagtgatt tggtaattta gtgccggaaa gattagtgcg 1412 gtgtcgcaga gtgatttggtaatttagtgc cggaaagatt tctttgtttt gaggagatct 1472 ttcgcgggac caaagggaggggggcagtgt aaagacgaca gaacaagcgt gaaggcctcg 1532 agagtcgaga cctcacagggtaccgcctag cgcctactgg ggtgaaagtg aagtcaagga 1592 gtcgggaggg tgtgtgtgaataccgtttgt agcagctagt gcgtccgtct gtcttttttt 1652 ttttctttct gtttattaattattaatagc ctgctagatt tcatttaaaa aaaaaaaaaa 1712 aaaaaaaaaa aaaaaaaaaaaa 1734 22 349 PRT Zea mays 22 Met Gly Asp Ala Ala Ala Ser Thr Ser AlaPro Thr Thr Pro Thr Ser 1 5 10 15 Ile Leu Ile Cys Leu Glu Asp Gly SerAsp Leu Leu Ala Asp Ala Asp 20 25 30 Asp Gly Ala Gly Thr Asp Leu Val ValAla Arg Asp Glu Arg Leu Leu 35 40 45 Val Val Asp Gln Asp Glu Glu Tyr ValAla Leu Leu Leu Ser Lys Glu 50 55 60 Ser Ala Ser Gly Gly Gly Gly Pro ValGlu Glu Met Glu Asp Trp Met 65 70 75 80 Lys Ala Ala Arg Ser Gly Cys ValArg Trp Ile Ile Lys Thr Thr Ala 85 90 95 Met Phe Arg Phe Gly Gly Lys ThrAla Tyr Val Ala Val Asn Tyr Leu 100 105 110 Asp Arg Phe Leu Ala Gln ArgArg Val Asn Arg Glu His Ala Trp Gly 115 120 125 Leu Gln Leu Leu Met ValAla Cys Met Ser Leu Ala Thr Lys Leu Glu 130 135 140 Glu His His Ala ProArg Leu Ser Glu Leu Pro Leu Asp Ala Cys Glu 145 150 155 160 Phe Ala PheAsp Arg Ala Ser Val Leu Arg Met Glu Leu Leu Val Leu 165 170 175 Gly ThrLeu Glu Trp Arg Met Val Ala Val Thr Pro Phe Pro Tyr Ile 180 185 190 SerCys Phe Ala Ala Arg Phe Arg Gln Asp Glu Arg Arg Ala Val Leu 195 200 205Val Arg Ala Val Glu Cys Val Phe Ala Ala Ile Arg Ala Met Ser Ser 210 215220 Val Glu Tyr Gln Pro Ser Thr Ile Ala Val Ala Ser Ile Leu Val Ala 225230 235 240 Arg Gly Arg Glu Thr Pro Ala Gly Asn Leu Asp Ala Leu Lys AlaIle 245 250 255 Leu Gly Ser Ser Phe Pro Gln Leu Asp Thr Gly His Val TyrSer Cys 260 265 270 Tyr Ser Ala Met Ile Arg Glu Asp Asp Lys Ser Pro ThrGln Ser Thr 275 280 285 Ser Thr Ser Thr Gly Val Ala Ser Ser Gly Val SerVal Ala Ala His 290 295 300 Ala Gly Gly Ser Gly Ser Pro Ser Pro Pro GlyAla Ser Val Ser Val 305 310 315 320 Gly Ala Asn Asn Ala Ala Gly Thr AlaPro Pro Ala Thr Thr Asp Asn 325 330 335 Arg Asn Lys Arg Arg Arg Leu ArgSer Pro Gln Arg Gln 340 345 23 21 DNA Zea mays primer_bind (1)...(21) 23cagtaccccc acgctgcaca g 21 24 26 DNA Zea mays primer_bind (1)...(26) 24tcacgcttgt tctgtcgtct ttacac 26 25 24 DNA Zea mays primer_bind(1)...(24) 25 gctgctgcaa gtccgcaacc actg 24 26 25 DNA Zea maysprimer_bind (1)...(25) 26 cgcttgttct gtcgtcttta cactg 25 27 27 DNA Zeamays primer_bind (1)...(27) 27 acctccatcc tcatctgcct ggaagac 27 28 24DNA Zea mays primer_bind (1)...(24) 28 ctggactgca ctgcactgca atgc 24 2925 DNA Zea mays primer_bind (1)...(25) 29 catcctcatc tgcctggaag acggc 2530 23 DNA Zea mays primer_bind (1)...(23) 30 aatgcactgc cagcagctga gct23

What is claimed is
 1. A method for increasing transformation frequencyin a plant cell comprising introducing into the plant cell a Cyclin D(CycD) polypeptide having transformation frequency-increasing activity.2. A method for increasing transformation frequency comprisingintroducing a polynucleotide of interest into a plant cell containing aCycD polypeptide having transformation frequency-increasing activity. 3.A method for increasing transformation frequency of a plant cellcomprising introducing into a plant cell an isolated CycD polynucleotidewherein said CycD polynucleotide is operably linked to a promotercapable of driving expression in the plant cell and wherein a CycDpolypeptide is expressed from said CycD polynucleotide, said CycDpolypeptide having transformation frequency-increasing activity.
 4. Amethod for increasing transformation frequency of a plant cellcomprising introducing into a plant cell an isolated CycD polynucleotidewherein said CycD polynucleotide is operably linked to a promotercapable of driving expression in said plant cell and wherein a CycDpolypeptide is expressed from said CycD polynucleotide, said CycDpolypeptide comprising cell cycle modulating activity.
 5. The method ofclaim 4 further comprising introducing into the plant cell at least onepolynucleotide of interest, said polynucleotide optionally beingoperably linked to a promoter capable of driving expression in saidplant cell.
 6. The method of claim 4 wherein said CycD polynucleotideencodes a plant CycD polypeptide.
 7. The method of claim 6 wherein saidplant CycD polypeptide is a monocot CycD polypeptide.
 8. The method ofclaim 7 wherein said monocot CycD is a maize CycD polypeptide.
 9. Themethod of claim 8 wherein said maize CycD is a CycDa or CycDcpolypeptide.
 10. The method of claim 9, wherein said CycD polypeptidehas 90% sequence identity to SEQ ID NOs 2 or
 14. 11. The method of claim10, further comprising a plant CycD specific binding motif, LXCXE and aconserved tryptophan residue in a conserved motif, WILKV.
 12. The methodof claim 4 wherein said CycD polynucleotide has 85% sequence identity toSeq ID Nos 1 or
 13. 13. The Cyc D polynucleotide of claim 4 wherein thepromoter is a constitutive promoter.
 14. The CycD polynucleotide ofclaim 4 wherein the promoter is inducible, tissue specific,developmentally regulated, or temporally regulated.
 15. The method ofclaim 4 wherein said CycD polynucleotide operably linked to a promoteris comprised in a recombinant expression cassette.
 16. The method ofclaim 4, wherein the plant cell is stably transformed.
 17. The method ofclaim 4, wherein the plant cell is a monocot or a dicot cell.
 18. Themethod of claim 17, wherein said monocot or dicot cell is from corn,soybean, wheat, rice, alfalfa, sunflower, or canola.
 19. The method ofclam 16, further comprising growing the plant cell into a plant.
 20. Themethod of claim 4, wherein increasing the level of CycD polypeptideexpression increases the number of transgenic events.
 21. A method ofincreasing transformation frequency in a plant cell comprisingintroducing into the plant cell a CycD RNA or polypeptide, wherein saidpolypeptide comprises cell cycle modulating activity.
 22. A method ofmodulating the cell cycle comprising transforming the plant cell with arecombinant expression cassette comprising an isolated CycDpolynucleotide operably linked to a promoter, wherein said CycDpolynucleotide encodes a CycD polypeptide which modulates cell cycleactivity.
 23. A method for providing a means of positive selectioncomprising expressing a CycD polypeptide in a plant cell and selectingfor cells exhibiting a positive growth advantage, said polypeptidehaving transformation frequency increasing activity.
 24. The method ofclaim 23 further comprising a polynucleotide of interest.
 25. The methodof claim 1, wherein the cell comprises said CycD polypeptide fromintroduced RNA or protein or protein delivered via Agrobacterium.
 26. Amethod of modulating the level of CycD protein in a cell, comprising:(a) transforming a cell with a recombinant expression cassettecomprising a CycD polynucleotide operably linked to a promoter; (b)growing the cell under cell-growing conditions for a time sufficient toinduce expression of the polynucleotide sufficient to modulate CycDprotein in the cell.
 27. The method of claim 26, wherein CycD protein isincreased.
 28. The method of claim 26, wherein CycD protein isdecreased.
 29. The method of claim 26, wherein the level of CycD proteinin the cell is transiently modulated by introducing CycD ribonucleicacid.
 30. The method of claim 26, wherein the CycD protein is present inan amount sufficient to alter cell division.
 31. The method of claim 26,wherein the CycD protein is present in an amount sufficient to increasethe number of dividing cells.
 32. The method of claim 26, wherein theCycD protein is present in an amount sufficient improves transformationfrequencies.
 33. The method of claim 26, wherein the CycD protein ispresent in an amount sufficient to alter cell growth.
 34. The method ofclaim 26, wherein the CycD protein is present in an amount sufficient toprovide a positive growth advantage for the cell.
 35. The method ofclaim 26, wherein the CycD protein is present in an amount sufficient toincrease the growth rate.
 36. The method of claim 26, wherein the cellis a plant cell and the plant cell is grown under conditions appropriatefor regenerating a plant capable of expressing CycD protein.
 37. Themethod of claim 36, wherein the plant cell is from corn, soybean, wheat,rice, alfalfa, sunflower, or canola.
 38. The method of claim 36, whereinthe CycD protein is present in an amount sufficient to increase cropyield.
 39. The method of claim 36, wherein the CycD protein is presentin an amount sufficient to alter plant height or size.
 40. The method ofclaim 36, wherein the CycD protein is present in an amount sufficient toenhance or inhibit organ growth.
 41. The method of claim 39, wherein theorgan is a seed, root, shoot, ear, tassel, stalk, pollen, or stamen. 42.The method of claim 40, wherein the level of CycD protein is modulatedto produce organ ablation.
 43. The method of claim 36, wherein the levelof CycD protein is modulated to produce parthenocarpic fruits.
 44. Themethod of claim 36, wherein the level of CycD protein is modulated toproduce male sterile plants.
 45. The method of claim 42, wherein theCycD protein is present in an amount sufficient to enhance embryogenicresponse.
 46. The method of claim 36, wherein the CycD protein ispresent in an amount sufficient to increase callus induction.
 47. Themethod of claim 36, wherein the level of CycD protein is modulated toprovide for positive selection.
 48. The method of claim 36, wherein thelevel of CycD protein is modulated to increase plant regeneration. 49.The method of claim 42, wherein the level of CycD protein is modulatedto alter the percent of time that the cells are arrested in G1 or G0phase.
 50. The method of claim 36, wherein the level of CycD protein ismodulated to alter the amount of time the cell spends in a particularcell cycle.
 51. The method of claim 43, wherein the level of CycDprotein is modulated to improve the response of the cells toenvironmental stress including dehydration, heat, or cold.
 52. Themethod of claim 36, wherein the level of CycD protein is modulated toincrease the number of pods per plant.
 53. The method of claim 36,wherein the level of CycD protein is modulated to increase the number ofseeds per pod or ear.
 54. The method of claim 36, wherein the level ofCycD protein is modulated to alter the lag time in seed development. 55.The method of claim 36, wherein the level of CycD protein is modulatedto provide hormone independent cell growth.
 56. The method of claim 26,wherein the level of CycD protein is modulated to increase the growthrate of cells in bioreactors.
 57. The method of claim 26, wherein thelevel of CycD protein in cells is: transiently modulated by introducingCycD ribonucleic acid.
 58. A method for transiently modulating the levelof CycD protein in plant cells comprising introducing CycD polypeptides.